Mirnas as therapeutic targets in cancer

ABSTRACT

MicroRNAs (miRNAs) are a class of non-coding small RNA molecules that regulate gene expression at the post-transcriptional level by interacting with 3′ untranslated regions (UTRs) of their target mRNAs. The invention relates to the application of miR-192 and miR-215. Both of these miRNAs impact cellular proliferation through the p53-miRNA circuit, and interact with dihydrofolate reductase (DHFR) and thymidylate synthase (TS). Particularly, upregulation of these miRNAs reduces cellular proliferation. The invention relates to this discovery. For example, inhibiting miR-192 and/or miR-215 sensitizes a neoplasm or a subject with a neoplasm to chemotherapeutic agents. Furthermore, measuring the levels of miR-192 and/or miR-215 provides one with information regarding whether the neoplasm or subject will respond to chemotherapeutic agents. Accordingly, the invention relates to composition and methods relating to the identification, characterization and modulation of the expression of miR-192 and miR-215.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 12/996,249 which was filed Mar. 15, 2011, which is the United Statesnational phase of International Application No. PCT/US2009/046353 filedJun. 5, 2009, which claims the benefit of U.S. Provisional ApplicationNo. 61/122,377 which was filed on Dec. 13, 2008 and U.S. ProvisionalApplication No. 61/059,197 which was filed Jun. 5, 2008. The disclosureof each of these documents is hereby incorporated in its entirety byreference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

The invention was made using U.S. Government funds, and therefore theU.S. Government has rights in the invention.

FIELD OF THE INVENTION

The present invention relates to compositions and methods relating tothe identification and characterization of genes and biological pathwaysrelated to these genes as represented by the expression of theidentified genes, as well as use of microRNAs (miRNAs) related to such,for therapeutic, prognostic, and diagnostic applications, particularlythose methods and compositions related to assessing and/or identifyingpathological conditions directly or indirectly related to miR-192 ormiR-215.

BACKGROUND OF THE INVENTION

Dihydrofolate reductase (DHFR) and Thymidylate Synthase (TS) are two keyenzymes for DNA synthesis and represent some of the most importanttargets for cancer chemotherapy. DHFR catalyzes the reduction of folateand 7, 8 dihydrofolate to 5, 6, 7, 8 tetrahydrofolate, the latter as theone-carbon donor is essential for the formation of thymidylate (dTMP)which is the precursor for DNA synthesis (see Banerjee et al., “Novelaspects of resistance to drugs targeted to dihydrofolate reductase andthymidylate synthase, Biochim. Biophys. Acta. (2002) 1587: 164-173). TScatalyzes the reductive methylation of dUMP by CH2H4folate to producedTMP and H2folate (see Carreras et al., “The catalytic mechanism andstructure of thymidylate synthase,” Annu. Rev. Biochem. (1995) 64:721-762). DHFR inhibitors, such as methotrexate (MTX), and TSinhibitors, such as 5-fluorouracil (5-FU) and Tomudex or Ralitrexed(TDX, ZD1694), are widely used chemotherapeutic drugs for the treatmentof osteosarcoma and colon cancer (see Widemann et al., “Understandingand managing methotrexate nephrotoxicity,” Oncologist. (2006) 11:694-703; see also Calvert, “An overview of folate metabolism: featuresrelevant to the action and toxicities of antifolate anticancer agents,”Semin. Oncol. (1999) 26: 3-10).

As such, TS and DHFR are the major targets of cancer chemotherapy in theclinic today. TDX, the third-generation TS inhibitor, is an active agentin the treatment of human colon and breast cancer (see Drake et al.,“Resistance to tomudex (ZD1694): multifactorial in human breast andcolon carcinoma cell lines,” Biochem. Pharmacol. (1996) 51: 1349-1355).The inhibitor of DHFR, such as MTX, is widely used in the treatment ofhuman leukemia, osteosarcoma and choriocarcinoma. Increased DHFR proteinlevels are reported to be associated with drug resistance (see Banerjeeet al., “Novel aspects of resistance to drugs targeted to dihydrofolatereductase and thymidylate synthase,” Biochem. Biophys. Acta. (2002)1587: 164-173), and low tumor expression levels of TS have also beenlinked with improved outcome for colon cancer patients treated with 5-FUchemotherapy (see Soong et al., “Prognostic significance of thymidylatesynthase, dihydropyrimidine dehydrogenase and thymidine phosphorylaseprotein expression in colorectal cancer patients treated with or without5-fluorouracil-based chemotherapy,” Ann. Oncol. (2008) 19: 915-919).However, MTX has the highest activity at the time when DNA synthesis,DHFR activity, DHFR content, and DHFR mRNA content increased and thelowest activity at the time when they decreased (see Yamauchi et al.,“Ohdo S. Cell-cycle-dependent pharmacology of methotrexate in HL-60,” J.Pharmacol. Sci. (2005) 99: 335-341).

Thus, there exists a need for better prognostic and diagnostic measures,treatment and control of neoplasm through application of small moleculesto target cells to affect various cellular components, such as TS andDHFR involved in cellular proliferation of neoplasia.

SUMMARY OF THE INVENTION

The invention provides an isolated inhibitory nucleic acid molecule thatis complementary to at least a portion of miR-192 (SEQ ID NO:1) ormiR-215 (SEQ ID NO:9). The inhibitory nucleic acid molecule decreasesthe expression of the at least a portion of miR-192 or miR-215 in acell. In an embodiment, the nucleic acid molecule is an antisensenucleic acid molecule. The antisense nucleic acid molecule has at leastan 85% sequence identity to the portion of miR-192 (SEQ ID NO:1),miR-215 (SEQ ID NO:9). In another embodiment, the portion has a sequenceselected from the group consisting of SEQ ID NOs:2, 3 and 10. In thisembodiment, the inhibiting nucleic acid molecule has at least 85%sequence identity to the sequence. In another embodiment, the nucleicacid molecule has a sequence consisting essentially of SEQ ID NOs:1-3 or9-10. In another embodiment, an expression vector comprises theinhibitory nucleic acid molecule. The inhibitory nucleic acid moleculemay be operably linked to a promoter suitable for expression in amammalian cell. The vector may be a viral vector. In another embodiment,a cell comprises the vector.

The invention further provides an isolated inhibitory nucleic acidmolecule that corresponds to at least a portion of miR-192 (SEQ ID NO:1)or miR-215 (SEQ ID NO:9). The inhibitory nucleic acid molecule decreasesthe expression of the miR-192 (SEQ ID NO:1) or miR-215 (SEQ ID NO:9) ina cell. In one embodiment, the portion has a sequence selected from thegroup consisting of SEQ ID NOs:2, 3 and 10. In another embodiment, thenucleic acid molecule has a sequence consisting essentially of SEQ IDNOs:1-3 or 9-10. In another embodiment, the inhibiting nucleic acidmolecule has at least 85% sequence identity to the portion of SEQ IDNOs:1 or 9, or at least 85% sequence identity to SEQ ID NOs:2, 3 or 10.The nucleic acid molecule is also an shRNA or an siRNA. In anembodiment, the nucleic acid molecule comprises at least onemodification. The modification may be a non-natural internucleotidelinkage, a backbone modification, or a substituted sugar moiety. Inanother embodiment, an expression vector comprises the inhibitorynucleic acid molecule. The inhibitory nucleic acid molecule may beoperably linked to a promoter suitable for expression in a mammaliancell. The vector may be a viral vector. In another embodiment, a cellcomprises the vector.

The invention further provides an isolated inhibitory nucleic acidmolecule that corresponds to a portion of a miR-192 promoter that bindsto a p53 binding sequence (SEQ ID NO: 25). The inhibitory nucleic acidmolecule comprises or consists essentially of a sequence or acomplementary sequence of SEQ ID NO:25, a fragment thereof, or a variantthereof. In this embodiment, the inhibitory nucleic acid molecule has atleast 85% sequence identity to SEQ ID NO: 25 or a fragment thereof.

Methods for delivery of the inhibitory nucleic acid molecules include,but are not limited to, using a delivery system such as viral vectors,liposomes, polymers, microspheres, gene therapy vectors, naked DNAvectors, carbon nanotubes and chemical linkers. One of ordinary skill inthe art would recognize other methods of delivering the inhibitorynucleic acid molecules into the cell or subject.

The invention further provides a method of modulating expression of acomponent of a cell, comprising contacting the cell with a nucleic acidcomprising a portion of a sequence or a complementary sequence selectedfrom an miR-192 sequence (SEQ ID NO:1), an miR-215 nucleic acid sequence(SEQ ID NO:9), miR-192 promoter binding site for p53 (SEQ ID NO:25), anantisense miR-192 sequence, an antisense miR-215 sequence and anantisense to the miR-192 promoter binding site for p53 in an amountsufficient to modulate the cellular component. In an embodiment, thenucleic acid is an antisense nucleic acid. The nucleic acid may be ansiRNA or an shRNA. In an embodiment, the cellular component is miR-192or miR-215. In another embodiment, the portion has a sequence selectedfrom the group consisting of SEQ ID NOs:2, 3, 10, antisense SEQ ID NO: 2and antisense SEQ ID NO: 3 and an antisense SEQ ID NO: 10. In anotherembodiment, the nucleic acid molecule has a sequence consistingessentially of SEQ ID NOs:1-3, 9-10, 25 or antisense sequences thereof.In another embodiment, the cellular component is p21 or p53. In anotherembodiment, the cellular component is regulated by p53. In anotherembodiment, the cellular component is DHFR or TS.

The invention further provides a method of modulating proliferation of acell, comprising contacting the cell with a nucleic acid having asequence or a complementary sequence to at least a portion of miR-192(SEQ ID NO:1), miR-215 (SEQ ID NO:9), a p53 region that binds to amiR-192 promoter or the miR-192 promoter that binds to p53 (SEQ IDNO:25) in an amount effective to modulate proliferation of the cell. Inanother embodiment, the nucleic acid is an antisense nucleic acid. Inanother embodiment, the portion has a sequence selected from the groupconsisting of SEQ ID NOs:2, 3, 10 and 25. In another embodiment, thenucleic acid molecule has a sequence consisting essentially of SEQ IDNOs:1-3, 9-10 or 25 or an antisense sequence thereof. In anotherembodiment, the nucleic acid is an siRNA or an siRNA. In anotherembodiment, the cell is a cancer stem cell. In another embodiment, thecell is a neoplastic cell. In another embodiment, the method ofmodulating proliferation of a cell is a method of increasing theproliferation of a cell.

The invention further provides a method of increasing the sensitivity ofa cell to a chemotherapeutic agent, comprising contacting the cell witha nucleic acid complementary to at least a portion of miR-192 (SEQ IDNO:1), miR-215 (SEQ ID NO:9) an miR-192 promoter binding site for p53(SEQ ID NO:25) or a p53 region that binds to the miRNA-192 promoter, inan amount effective to sensitize the cell to the chemotherapeutic agent.Examples of chemotherapeutic agents include, but are not limited to,methotrexate, fluorouracil (5-FU), nolatrexed, ZD9331, GS7904L andralitrexed. In another embodiment, the portion has a sequence selectedfrom the group consisting of SEQ ID NOs:2, 3, 10 and 25. In anotherembodiment, the nucleic acid molecule has a sequence consistingessentially of SEQ ID NOs:1-3, 9-10, and 25. In embodiments, the nucleicacid is an antisense nucleic acid. In another embodiment, the nucleicacid is an siRNA or an shRNA. In another embodiment, the cell is acancer stem cell. In another embodiment, the cell is a neoplastic cell.

The invention further provides a method of increasing the sensitivity ofa cell to radiation, comprising contacting the cell with a nucleic acidcomplementary to at least a portion of miR-192 (SEQ ID NO:1), miR-215(SEQ ID NO:9) an miR-192 promoter binding site for p53 (SEQ ID NO:25) ora p53 region that binds to the miRNA-192 promoter, in an amounteffective to sensitize the cell to radiation. In embodiments, thenucleic acid is an antisense nucleic acid. In another embodiment, thenucleic acid is an siRNA or an shRNA. In another embodiment, the portionhas a sequence selected from the group consisting of SEQ ID NOs:2, 3 and10. In another embodiment, the nucleic acid molecule has a sequenceconsisting essentially of SEQ ID NOs:1-3, 9-10, and 25. In anotherembodiment, the cell is a cancer stem cell. In another embodiment, thecell is a neoplastic cell.

The invention further provides a method of treating a neoplasm in asubject, comprising administering to the subject an effective amount ofa nucleic acid molecule that inhibits expression of miR-192 or miR-215,and a second therapy, wherein inhibition of expression of miR-192 ormiR-215 sensitizes the neoplasm to the second therapy. In anotherembodiment, the second therapy comprises administering achemotherapeutic agent. In one embodiment, the chemotherapeutic agent isselected from the group consisting of a DHFA inhibitor and examples ofchemotherapeutic agents include, but are not limited to, methotrexate,fluorouracil (5-FU), nolatrexed, ZD9331, GS7904L and ralitrexed. Inanother embodiment, the second therapy comprises administering radiationto the subject. The neoplasm may be cancer. The cancer may be selectedfrom the group consisting of colon cancer, pancreatic cancer, lungcancer, breast cancer, cervical cancer, gastric cancer, kidney cancer,leukemia, liver cancer, lymphoma, ovarian cancer, prostate cancer,rectal cancer, sarcoma, skin cancer, testicular cancer, and uterinecancer.

The invention further provides a method of diagnosing a neoplasm in asubject comprising determining the level of expression of miR-192 ormiR-215. In one embodiment, the method of diagnosing the neoplasm in thesubject comprises determining the level of expression of miR-192 andmiR-215.

The invention further provides a method of identifying a neoplasmresistant to chemotherapy comprising determining the level of expressionin the neoplasm of miR-192 or miR-215, and identifying the neoplasm asresistant to therapy if the level of the miR-215 is elevated or thelevel of miR-192 is reduced as compared to a control. In one embodimentthe method of identifying the neoplasm resistant to chemotherapycomprises determining the levels of miR-192 and miR-215, and identifyingthe neoplasm as resistant to therapy if the levels of miR-192 andmiR-215 are elevated.

The invention further provides a method of determining whether aneoplasm is a candidate for treatment with a chemotherapeutic agentcomprising evaluating the level of expression of an miRNA, wherein themiRNA is miR-192 or miR-215, and rejecting the candidate if theexpression of the miRNA is elevated; or accepting the candidate if theexpression of the miRNA is reduced. In one embodiment, the miRNA ismiR-192 and miR-215. In another embodiment, the rejected candidate wouldbe a candidate for the methods of increasing sensitivity or treating aneoplasm discussed herein.

The invention further provides a kit for analysis of a pathologicalsample. The kit comprising in a suitable container an miRNAhybridization reagent for determining the level of miR-192 or miR-215.In an embodiment, the mRNA hybridization reagent comprises ahybridization probe. In another embodiment, the mRNA hybridizationreagent comprises amplification primers. In another embodiment, thehybridization probe or amplification primers complementary to a sequenceselected from the group consisting of SEQ ID NOs:1, 2, 3, 9, 10 andfragments thereof. The pathological sample can be any sample commonlytaken from a subject, such as, for example, blood, urine, tissue, orother bodily fluid.

The invention further provides a method of identifying an agent thatinhibits expression of an miRNA selected from the group consisting ofmiR-192 and miR-215, which comprises contacting a cell that expressesthe miRNA with an agent, and comparing the expression level of the miRNAin the cell contacted by the agent with the expression level of themiRNA in the absence of the agent, wherein the agent is an inhibitor ofthe miRNA if expression of the miRNA is reduced. In an embodiment, thetest cell overexpresses the miRNA.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts a predicted secondary structure of the interactionbetween miR-192 and the 3′-UTR region of DHFR mRNA.

FIG. 2A is a graph illustrating the expression of miR-192 in HCT-116(wt-p53) cells transfected with miR-192.

FIG. 2B is a graph illustrating the expression of miR-24 in HCT-116(wt-p53) cells transfected with miR-24.

FIG. 2C depicts an image from a Western immunoblot showing theexpression of DHFRT protein in HCT-116 (wt-p53) cells transfected withDHFR specific siRNA, miR-192 or miR-24.

FIG. 2D is a graph illustrating the expression of DHFR mRNA in HCT-116(wt-p53) cells transfected with DHFR specific siRNA, miR-192 or miR-24.

FIG. 3 is a graph illustrating the impact of miR-192 on cellproliferation with MTX treatment in HCT-116 (wt-p53) cells transfectedwith DHFR specific siRNA or miR-192.

FIGS. 4A-D is a graph illustrating the impact of miR-192 on cellproliferation in HCT-116 (wt-p53) cells (FIG. 4A), RKO cells (FIG. 4B),HCT-116 (null-p53) cells (FIG. 4C) and HT-29 (FIG. 4D) cells transfectedwith non-specific miR or miR-192.

FIGS. 5 A-D are graphs illustrating impact of miR-192 on cellproliferation in HCT-116 (wt-p53) (FIG. 5 A-B) and HCT-116 (null-p53)(FIG. 5 C-D) cells transfected with non-specific miR or miR-192.

FIGS. 6A-B are images from a Western immunoblot of p53, p21 and Baxexpression in HCT-116 (wt-p53) cells transfected with non-specific miR,DHFR siRNA or miR-192.

FIG. 7 is an image from a Western immunoblot for E2F3 and Rb expressionin HCT-116 (wt-p53) cells transfected with non-specific miR, DHFR siRNAor miR-192.

FIG. 8A is an image from a Western immunoblot for p53 expression inHCT-116 (wt-p53) cells, RKO (wt-p53) cells, HCT-116 (null-p53) cells andHT-29 cells after treatment with MTX.

FIG. 8B is a graph depicting the expression of endogenous mature miR-192in HCT-116 (wt-p53) cells, RKO (wt-p53) cells, HCT-116 (null-p53) cellsand HT-29 cells after treatment with MTX.

FIG. 9A is a diagram of miR-192 p53 binding site relative to miR-192 onchromosome 11.

FIG. 9B is a graph illustrating the immunoprecipitation qPCR analysisusing chromosome DNA isolated with non-specific a-tubulin, p53monoclonal antibody for the conserved p53 binding site located on themiR-192 or p21 promoter.

FIG. 10 shows inhibition of cell proliferation in either HCT-116(wt-p53) (Panel A) or U-2 OS (Panel B) cells transfected with miR-215precursors, i.e., a longer piece of miRNA (72 bps) with hairpin stemloop structure that is further processed by DICER to mature miRNA,compared with a non-specific miRNA control. The reduction in OD forcultures of HCT-116 (wt-p53) or U-2 OS cells were approximately 40% or58% at day 5 respectively.

FIG. 11 depicts proportions of cultured U-2 OS or MG63 cells, and U-2 OSor MG63 cells transfected with miR-192 or miR-215 at various stages ofthe cell cycle.

FIG. 12 depicts the levels of cell cycle control gene products p53 andp21 determined by Western immunoblot analysis.

FIG. 13 provides alignments of miR-215 with regions of DHFR and TS.

FIG. 14 depicts an analysis of levels of DHFR (Panel A) or TS (Panel B)protein in cells transfected with miR-215.

FIG. 15 depicts the levels of DHFR or TS mRNA in U-2 OS cellstransfected with miR-215.

FIG. 16 depicts the level of luciferase reporter activity from aconstruct containing the 3′ UTR binding sequence for miR-215 (or amutated binding sequence) in response to transfection with miR-215.

FIG. 17 shows that the effect of miR-192 (Panel A) or miR-215 (Panel B)on proliferation of HCT-116 (wt-p53) cells treated with methotrexate.

FIG. 18 depicts miR-192 and miR-215 expression in CD133⁺ HCT-116(wt-p53) cells as compared to CD133⁻ cells.

FIG. 19 depicts miR-215 expression in paired human colon cancer andcounterpart normal tissues.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have discovered that miR-192 and miR-215, individually,decrease the expression of TS and DHFR protein, and further found thatmiR-192 and miR-215, individually, change the sensitivity of cancerouscells such as, for example, HCT-116 (wt-p53) or U-2 OS cells to TDX orMTX. The inventors have found that down-regulation of TS or DHFR proteinby a siRNA specific against TS or DHFR increases the sensitivity of TDXor MTX in the colon cancer or osteosarcoma cell lines, whereas eventhough miR-192 and miR-215 also down-regulated the expression levels ofTS or DHFR, they did not increase the chemosensitivity of TDX or MTXcompared to a non-specific miRNA control. TDX or MTX are considered tobe more effective on the cells in the S-phase. As illustrated in FIGS. 5and 11, siRNAs specific against TS or DHFR do not decrease the cells inthe S-phase, whereas the cells in the S-phase were reduced in themiR-192 transfected cells and miR-215 transfected cells. Thus,down-regulating miR-192 or miR-215 increases the sensitivity of a cellor a subject to chemotherapy.

The inventors have found that colon cancer stem cells depend on, atleast in part, elevated miR-192 or miR-215 to have a reduced cellproliferation phenotype. The advantage of tumor stem cells using miR-192or miR-215 to achieve this is that translational control by miR-192 ormiR-215 is an acute response, readily reversible without permanentlydegrading its target mRNAs such as TS and DHFR or trigger apoptosis.This also suggests why half of the colon cancer cases containing wildtype p53 are still resistant to chemotherapeutic treatment. Thismechanism also provides a novel approach to selectively killing coloncancer stem cells by inhibiting miR-192 or miR-215 and subsequentlyeliminating them with chemotherapeutic agents. Furthermore, thismechanism also provides a novel approach for identifying a candidate whowill respond to chemotherapeutic treatment by inhibiting miR-192 and/ormiR-215.

In certain aspects, the invention is directed to methods for theassessment, analysis, and/or therapy of a cell or subject where certaingenes have a reduced or increased expression (relative to normal) as aresult of an increased or decreased expression of miR-192 or miR-215.The expression profile and/or response to miR-192 or miR-215 expressionor inhibition may be indicative of a disease or an individual with apathological condition such as, for example, cancer.

According to a first embodiment, the miR-192 or miR-215 inhibitors mayinclude antisense nucleic acids or molecules. Antisense nucleic acidsare effective in inhibiting human miRNAs. Antisense nucleic acidsinclude non-enzymatic nucleic acid compounds that bind to a targetnucleic acid by, for example, RNA-RNA, RNA-DNA or RNA-PNA interactionsand effect the target nucleic acid. Generally, these molecules arecomplementary to a target sequence along a single contiguous sequence ofthe antisense nucleic acid. In this embodiment, the antisense nucleicacid reduces expression of miR-192 or miR-215.

In another embodiment, the inhibitors include fragments of the nucleicacid molecules that bind to miR-192 (SEQ ID NO:1) or miR-215 (SEQ IDNO:9), bind to an miR-192 promoter binding sequence for p53 (SEQ IDNO:25), or bind to the p53 sequence that is complementary to the miR-192promoter. A suitable fragment can be at least 8, at least 10, at least12, at least 14, at least 16, at least 18, at least 19, at least 20, atleast 21, at least 22, at least 23, at least 24, or at least 25nucleotides in length. Non-limiting examples of suitable fragmentsinclude nucleic acids having sequences complementary to SEQ ID NOs:2, 3,9 or fragments thereof. One of ordinary skill in the art recognizes thatnucleic acids complimentary to other portions of SEQ ID NO:1 or SEQ IDNO:9 would be equally effective.

In another embodiment, antisense nucleic acids may also bind to asubstrate nucleic acid and form a loop. In this embodiment, theantisense nucleic acids may be complementary to two or morenon-contiguous substrate sequences and/or two or more non-contiguoussequence portions of an antisense nucleic acid may be complementary to atarget sequence.

In another embodiment, antisense nucleic acids may be complementary to aguide strand of an miRNA positioned in the RNA silencing complex. Inanother embodiment, antisense nucleic acids may be used to target anucleic acid by means of DNA-RNA interactions. In this embodiment, RNaseH is activated to digest the target nucleic acid as would be understoodby one of ordinary skill in the art. For example, the antisense nucleicacids may comprise one or more RNase H activating region capable ofactivating RNase H to cleave a target nucleic acid. The RNase Hactivating region may comprise any suitable backbone. For example, inthis embodiment, the RNase H activating region may comprise aphosphodiester, phosphorothioate, phosphorodithioate, 5′-thiophosphate,phosphoramidate and/or methylphosphonate.

In another embodiment, the nucleic acid molecule may comprise one ormore modifications. Antisense nucleic acids according to the embodimentsmay comprise natural-type oligonucleotides and modifiedoligonucleotides. For example, in this embodiment, the antisense nucleicacid may comprise phosphorothioate-type oligodeoxyribonucleotides,phosphorodithioate-type oligodeoxyribonucleotides,methylphosphonate-type oligodeoxyribonucleotides, phosphoramidate-typeoligodeoxyribonucleotides, H-phosphonate-type oligodeoxyribonucleotides,triester-type oligodeoxyribonucleotides, alpha-anomer-typeoligodeoxyribonucleotides, peptide nucleic acids, locked nucleic acids,and nucleic acid-modified compounds. It will be readily apparent to oneof ordinary skill in the art that other oligonucleotides are within thescope and spirit of this invention.

In another embodiment, the modification may comprise internucleosidelinkages. For example, an inhibitory nucleic acid may be based onT-modified oligonucleotides containing oligodeoxynucleotide gaps withinternucleotide linkages modified to phosphorothioates for nucleaseresistance. The presence of methylphosphonate modifications increasesthe affinity of the oligonucleotide for its target RNA and thusincreases its effectiveness in inhibiting the target RNA. Thismodification also increases the nuclease resistance of the modifiedoligonucleotide.

In another embodiment, the modification may comprise a backbonemodification. For example, oligomers having modified backbones mayinclude those that retain a phosphorus atom in the backbone and thosethat do not have a phosphorus atom in the backbone. For example,nucleobase oligomers that have modified oligonucleotide backbonesinclude, but are not limited to, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkyl-phosphotriesters, methyl and other alkyl phosphonates,phosphinates, phosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates. Other forms, including, but not limited to, salts,mixed salts and free acid forms, are also contemplated.

Nucleobase oligomers having modified oligonucleotide backbones that donot include a phosphorus atom therein have backbones that are formed byshort chain alkyl or cycloalkyl internucleoside linkages, mixedheteroatom and alkyl or cycloalkyl internucleoside linkages, or one ormore short chain heteroatomic or heterocyclic internucleoside linkages.These include, but are not limited to, those having morpholino linkages,siloxane backbones, sulfide, sulfoxide and sulfone backbones, formacetyland thioformacetyl backbones, methylene formacetyl and thioformacetylbackbones, alkene containing backbones, sulfamate backbones,methyleneimino and methylenehydrazino backbones, sulfonate andsulfonamide backbones, and/or amide backbones.

In another embodiment, the modification may also comprise one or moresubstituted sugar moieties. For example, the RNase H activating regionmay comprise deoxyribose, arabino and/or fluoroarabino nucleotide sugarchemistry. Such modifications may also include 2′-O-methyl and2′-methoxyethoxy modifications, 2′-dimethylaminooxyethoxy,2′-aminopropoxy and 2′-fluoro, and modifications at other positions onthe oligonucleotide or other nucleobase oligomer, particularly the 3′position of the sugar on the 3′ terminal nucleotide. Nucleobaseoligomers may also have sugar mimetics. In another embodiment, both thesugar and the internucleoside linkage may be replaced with novel groups.The nucleobase units are maintained for hybridization with at least aportion of miR-192 (SEQ ID NO:1), miR-215 (SEQ ID NO:9), an miR-192promoter sequence that binds to p53 (SEQ ID NO:25) or the p53 sequencethat binds to the miR-192 promoter (SEQ ID NO:25).

Those skilled in the art will recognize that the foregoing arenon-limiting examples and that any combination of phosphate, sugar andbase chemistry of a nucleic acid that supports the activity of RNase Henzyme is within the scope of the present invention.

According to another embodiment, the invention relates to the use ofinterference RNA (RNAi) to alter the expression of miR-192 or miR-215.In one embodiment, the expression is altered by reducing the expressionof miR-192 or miR-215. In another embodiment, the expression of miR-192and miR-215 is altered. In another embodiment, the expression is alteredby reducing the expression of miR-192 and miR-215. RNAi comprises doublestranded RNA that can specifically block expression of a target gene.Double-stranded RNA (dsRNA) blocks gene expression in a specific andpost-transcriptional manner. RNAi provides a useful method of inhibitinggene expression in vitro or in vivo. RNAi can comprise either longstretches of dsRNA identical or substantially identical to the targetnucleic acid sequence or short stretches of dsRNA identical to orsubstantially identical to only a region of the target nucleic acidsequence.

In embodiments RNAi includes, but are not limited to, small interferingRNAs (siRNAs), small hairpin RNAs (shRNAs), and other RNA species, suchas non-enzymatic nucleic acids, which can be cleaved in vivo to formsiRNAs. In this embodiment, RNAi may also include RNAi expressionvectors capable of giving rise to transcripts which form dsRNAs orshRNAs in cells, and/or transcripts which can produce siRNAs in vivo.

The RNAi may comprise a nucleotide sequence that hybridizes underphysiologic conditions of the cell to the nucleotide sequence of atleast a portion of the RNA transcript for the target gene. These RNAihave the advantage of being able to tolerate variations in sequence thatmay arise from, for example, genetic mutation, strain polymorphism orevolutionary divergence. The number of tolerated nucleotide mismatchesbetween the target sequence and the RNAi sequence is no more than 1 in 5basepairs, or 1 in 10 basepairs, or 1 in 20 basepairs, or 1 in 50basepairs. Mismatches in the center of the siRNA duplex are mostcritical and may essentially abolish cleavage of the target RNA. Incontrast, nucleotides at the 3′ end of the siRNA strand that iscomplementary to the target RNA do not significantly contribute tospecificity of the target recognition. In this embodiment, the antisensenucleic acid molecule has at least 85% sequence identity to SEQ IDNOs:1-3, 9 or 10.

Sequence identity may be optimized by sequence comparison and alignmentalgorithms known in the art and calculating the percent differencebetween the nucleotide sequences. In this embodiment, the preferredsequence identity between the inhibitory RNA and the portion of thetarget gene is greater than 90%, 95%, 96%, 97%, 98%, 99% or 100%.Alternatively, the duplex region of the RNA may be defined functionallyas a nucleotide sequence that is capable of hybridizing under specifiedconditions with a portion of the target gene transcript.

The double-stranded structure may be formed by a singleself-complementary RNA strand or two complementary RNA strands. RNAduplex formation may be initiated either inside or outside the cell. TheRNA may be introduced in an amount which allows delivery of at least onecopy per cell. Higher doses of double-stranded material may yield moreeffective inhibition, while lower doses may also be useful for specificapplications. Inhibition is sequence-specific in that nucleotidesequences corresponding to the duplex region of the RNA are targeted forgenetic inhibition.

In this embodiment, the siRNAs are around 19-30 nucleotides long, andeven more preferably 21-23 nucleotides. The siRNAs effectively recruitnuclease complexes and guide the complexes to the target mRNA by pairingto the specific sequences. As a result, the target mRNA is degraded bythe nucleases in the protein complex. In embodiments, the 21-23nucleotides siRNA molecules comprise a 3′ hydroxyl group. In certainembodiments, the siRNA can be generated by the processing of longerdouble-stranded RNAs, for example, in the presence of the enzyme dicer.The siRNA molecules can be purified using a number of techniques knownto those of skill in the art such as, for example, gel electrophoresis,non-denaturing column chromatography, chromatography, glycerol gradientcentrifugation, and/or affinity purification with an antibody.

In this embodiment, the shRNAs can be synthesized exogenously or can beformed by transcribing from RNA polymerase III promoters in vivo.Examples of making and using such shRNAs for gene silencing in mammaliancells are known in the art. Preferably, such shRNAs are engineered incells or in an animal to ensure continuous and stable suppression of adesired gene. It is recognized in the art that siRNAs can be produced byprocessing a shRNA in the cell.

In another embodiment, the invention relates to the use of suitableribozyme molecules, such as, for example, RNA endoribonucleases andhammerhead ribozymes, designed to catalytically cleave mRNA transcriptsto prevent translation of mRNA. Hammerhead ribozymes cleave mRNAs atlocations dictated by flanking regions that form complementary basepairs with the target mRNA, which have a base sequence of 5′-UG-3′.

According to another embodiment, polynucleotide or expression vectortherapy for treating neoplasia featuring a polynucleotide encoding aninhibitory nucleic acid molecule or analog thereof that targets miR-192or miR-215 is provided. In this embodiment, the antisense nucleic acidmay cause inhibition of expression by hybridizing with miR-192 ormiR-215 and/or genomic sequences encoding miR-192 or miR-215. Expressionvectors encoding inhibitory nucleic acid molecules can be delivered tocells of a subject having a neoplasia in a form in which they can betaken up and expressed so that therapeutically effective levels may beachieved. The expression vector produces an oligonucleotide which iscomplementary to at least a unique portion of miR-192 or miR-215.Methods for delivery of the polynucleotides to the cell according to theinvention include, but are not limited to, using a delivery system suchas viral vectors, liposomes, polymers, microspheres, gene therapyvectors, naked DNA vectors, carbon nanotubes and chemical linkers. Oneof ordinary skill in the art would recognize other methods of deliveringpolynucleotides into the cell or subject. Nucleic acid probes may alsobe modified so that they are resistant to endogenous nucleases such as,for example, exonucleases and/or endonucleases, and are therefore stablein vivo.

Inhibitory nucleic acid molecule expression for use in polynucleotidetherapy methods can be directed from any suitable promoter and regulatedby any appropriate mammalian regulatory element. Promoters may include,but are not limited to, the human cytomegalovirus, simian virus 40,and/or metallothionein promoters. In this embodiment, enhancers known topreferentially direct gene expression in specific cell types can be usedto direct the expression of a nucleic acid. The enhancers used caninclude, without limitation, those that are characterized as tissue- orcell-specific enhancers.

Transducing viral vectors such as, for example, retroviral, adenoviral,lentiviral and adeno-associated viral vectors, can be used as expressionvectors for somatic cell gene therapy. Viral vectors are especiallyuseful because of their high efficiency of infection, and stableintegration and expression. In this embodiment, for example, apolynucleotide encoding an inhibitory nucleic acid molecule can becloned into a retroviral vector and expression can be driven from itsendogenous promoter, from the retroviral long terminal repeat, or from apromoter specific for a target cell type of interest. Other viralvectors that can be used include, for example, a vaccinia virus, abovine papilloma virus, or a herpes virus, such as Epstein-Barr Virus.

In another embodiment, the invention comprises an inhibitory nucleicacid molecule that corresponds to a portion of a miR-192 promoterbinding sequence for p53 (SEQ ID NO:25). The inhibitory nucleic acidmolecule comprises or consists essentially of a sequence or acomplementary sequence of SEQ ID NO:25, a fragment thereof or avariation thereof. The inventors have discovered that SEQ ID NO:25 is aportion of the miR-192 promoter (SEQ ID NO:8) that binds to p53.Accordingly, the expression of miR-192 can be altered by administeringan inhibitory nucleic acid molecule that comprises a sequence of SEQ IDNO:25, a fragment thereof, or a variation thereof. In this embodiment,the inhibitory nucleic acid molecule would bind to the p53 site at themiR-192 promoter binding site and thereby block the binding of p53 tothe miR-192 promoter. The expression of miR-192 can also be altered byadministering an inhibitory nucleic acid molecule that comprises asequence complementary to SEQ ID NO:25, a fragment thereof, or avariation thereof. In this embodiment, the inhibitory nucleic acidmolecule would bind to the miR-192 promoter thereby blocking p53 frombinding to the miR-192 promoter region and block transcription ofmiR-192.

The inhibitory nucleic acid molecule can consist essentially of asequence or a sequence complementary to SEQ ID NO:25, a fragmentthereof, or a variation. In such embodiments, the inhibitory nucleicacid molecule may contain other components that are not involved inbinding to the p53 binding sequence or the miR-192 promoter region.These components may include, but are not limited to, other nucleicacids, amino acids ligands, linkers, or other modification that will noteffect the primary function of the inhibitory nucleic acid molecule inthese embodiments, blocking the binding of p53 to the miR-192 promoterregion and inhibiting the transcription of miR-192. For example, theinhibitor nucleic acid molecule may contain other nucleotidescomplementary to a position of the miR-192 promoter (SEQ ID NO:8) thatit does not interact with the p53 binding sequence.

To effectively block the binding of p53 to the miR-192 promoter and thetranscription of miR-192, the full sequence or complementary sequence ofSEQ ID NOs:8 or 25 need not be utilized. A fragment of the sequence ofcomplementary sequence would be adequate to block the binding of p53 tothe miR-192 promoter and the transcription of miR-192. A suitablefragment can be at least 8, at least 10, at least 12, at least 14, atleast 16, at least 18, at least 19, at least 20, at least 21, at least22, at least 23, at least 24, or at least 25 nucleotides in length.

In Example 8 (see below), a three base pair gap between SEQ ID NO:25 andthe p53 binding is discussed. Accordingly, 100% homology is notnecessary. Instead, at least 85%, at least 90%, at least 92%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99% or 100% homology is sufficient to enable the inhibitory nucleic acidmolecule to bind to the miR-192 promoter or the p53 binding site.

In another embodiment, a non-viral approach may be employed for theintroduction of an inhibitory nucleic acid molecule therapeutic to acell of a patient diagnosed as having a neoplasia. For example, aninhibitory nucleic acid molecule that targets a miRNA-215 can beintroduced into a cell by administering the nucleic acid in the presenceof lipofection, asialoorosomucoid-polylysine conjugation, or bymicro-injection under surgical conditions. In this embodiment, theinhibitory nucleic acid molecules are administered in combination with aliposome and protamine. Gene transfer can also be achieved usingnon-viral means involving transfection in vitro. Such methods includethe use of calcium phosphate, DEAE dextran, electroporation, andprotoplast fusion. Liposomes can also be potentially beneficial fordelivery of DNA into a cell.

For any particular subject, the specific dosage regimes should beadjusted over time according to the individual need and the professionaljudgment of the person administering or supervising the administrationof the compositions.

Methods of modulating expression of cellular components in an amountsufficient to modulate the cellular component are also provided. Inembodiments, the cellular components may comprise miR-192 or miR-215,p21, p53, DHFR, TS or any cellular component regulated by thesecomponents. One of ordinary skill in the art would recognize that othercellular components may be modulated and are within the scope and spiritof this invention.

p53 and p21, a downstream target of the p53 pathway of growth control,are reported to block cells at G2 checkpoint mainly through inhibitingCdc2 activity, the cyclin-dependent kinase that normally drives cellsinto mitosis, which is the ultimate target of pathways that mediaterapid arrest in G2 in response to DNA damage as reported in, forexample, Taylor et al., “Regulation of the G2/M transition by p53”,Oncogene. (2001); 20: 1803-1815; Stark et al., “Control of the G2/Mtransition,” Mol. Biotechnol. (2006); 32: 227-248; and Bunz et al.,“Requirement for p53 and p21 to sustain G2 arrest after DNA damage”,Science. (1998); 282: 1497-1501.

The inventors have discovered that miR-192 or miR-215 can induceG2-arrest in HCT-116 (wt-p53) and U-2 OS cells. Transfection of miR-192or miR-215 precursor into HCT-116 (wt-p53) and U-2 OS cells to analyzethe mechanism of cell proliferation inhibition by miR-192 or miR-215indicate that over-expression of miR-192 or miR-215 led to a significantincrease of the p53 and p21 protein in both HCT-116 (wt-p53) and U-2 OScells. FIGS. 6 and 12 depicts an evaluation of the levels of cell cyclecontrol genes p53 and p21 by Western immunoblot analysis. As illustratedin FIGS. 6 and 12, miR-192 or miR-215 contributes to the inhibition ofcell proliferation at least partially by the induction of G2-arrest inHCT-116 (wt-p53) and U-2 OS cells, which was through over-expression ofG2-checkpoint genes p53 and p21.

The inventors have discovered that miR-192 or miR-215 suppresses cellproliferation. Since most miRNAs have more than one target range fromdozens to hundreds (see Wiemer, “The role of microRNAs in cancer: Nosmall matter”, Eur. J. Cancer. (2007) 43:1529-1544), miR-192 or miR-215most likely targets the enzymes for DNA biosynthesis, such as TS andDHFR, and also leads to the inhibition of cell proliferation in cancercells. As illustrated in FIG. 13, the inventors used the Sanger database(microrna.sanger.ac.uk) to identify TS and DHFR as the putative targetsof miR-192 and miR-215. As illustrated in FIGS. 2 and 14, at 48 h aftertransfection, the inventors extracted the proteins and searched for thechanges in TS or DHFR protein levels by Western immunoblot analysis.Oligofectamine alone and non-specific miRNA were used as the negativecontrols. The DHFR expression is down-regulated by miR-192. UsingmiR-192 as a positive control of DHFR down-regulation, theover-expression of miR-215 and miR-192 was confirmed by real timeqRT-PCR analysis using U6 RNA to normalize the expression (see FIG. 11).Introduction of miR-192 or miR-215 clearly decreases TS or DHFR proteinlevels (see FIG. 2 and FIG. 14, lanes 3 and 4). The inventors alsoanalyzed the expression level of TS or DHFR mRNA using real time qRT-PCRanalysis. As illustrated in FIG. 15, the inventors discovered that therewas no reduction in TS or DHFR mRNA expression by miR-192 or miR-215(column 3) and miR-192 (column 4). Thus, the suppression of TS or DHFRexpression was regulated at the translational level without thedegradation of TS or DHFR mRNA.

In another embodiment, a method of increasing proliferation of a cell isprovided using the mechanisms of the various pathways disclosed herein.In this embodiment, the cell is contacted with a nucleic acidcomplementary to at least a portion of miR-192 (SEQ ID NO:1), miR-215(SEQ ID NO:9), an miR-192 promoter sequence that binds to p53 (SEQ IDNO:25) or the p53 sequence that binds to the miR-192 promoter. Theamount of nucleic acid complementary to miR-192 (SEQ ID NO:1), miR-215(SEQ ID NO:9), an miR-192 promoter sequence that binds to p53 (SEQ IDNO:25) or the p53 sequence that binds to the miR-192 promoter (SEQ IDNO:25) effective to increase proliferation of the cell is notparticularly limited. In embodiments, the amount is in the range of morethan about 20% for cell proliferation and more than about 2-fold ofIC₅₀. In embodiments, the nucleic acid may comprise an antisense nucleicacid, siRNA or shRNA. In embodiments, the cell may comprise a cancerstem cell or a neoplastic cell.

In another embodiment, a method of increasing the sensitivity of a cellto a chemotherapeutic agent is provided using the mechanisms of thevarious pathways disclosed herein. In this embodiment, the cell iscontacted with a nucleic acid complementary to at least a portion ofmiR-192 (SEQ ID NO:1), miR-215 (SEQ ID NO:9), an miR-192 promotersequence that binds to p53 (SEQ ID NO:25) or the p53 sequence that bindsto the miR-192 promoter (SEQ ID NO:25). The amount of nucleic acidcomplementary to miR-192, (SEQ ID NO:1), miR-215 (SEQ ID NO:9), anmiR-192 promoter sequence that binds to p53 (SEQ ID NO:25) or the p53sequence that binds to the miR-192 promoter effective to sensitize thecell to the chemotherapeutic agent is not particularly limited. Inembodiments, the amount is in the range of more than about 20% for cellproliferation and more than about 2-fold of IC₅₀. In an embodiment, thechemotherapeutic agent is selected from the group consisting of a DHFAinhibitor and a TS inhibitor. Examples of chemotherapeutic agentinclude, but are not limited to, methotrexate (MTX), fluorouracil(5-FU), nolatrexed, ZD9331, GS7904L and ralitrexed (TDX). One ofordinary skill would recognize other chemotherapeutic agents useful inthis embodiment. In embodiments, the nucleic acid may comprise anantisense nucleic acid, siRNA or shRNA. In embodiments, the cell maycomprise a cancer stem cell or a neoplastic cell.

In another embodiment, a method of increasing the sensitivity of a cellto radiation is provided using the mechanisms of the various pathwaysdisclosed herein. In this embodiment, the cell is contacted with anucleic acid complementary to at least a portion of miR-192, (SEQ IDNO:1), miR-215 (SEQ ID NO:9), an miR-192 promoter sequence that binds top53 (SEQ ID NO:25) or the p53 sequence that binds to the miR-192promoter (SEQ ID NO:25). The amount of nucleic acid complementary tomiR-192 (SEQ ID NO:1), miR-215 (SEQ ID NO:9), an miR-192 promotersequence that binds to p53 (SEQ ID NO:25) or the p53 sequence that bindsto the miR-192 promoter effective to sensitize the cell to radiation isnot particularly limited. In embodiments, the amount is in the range ofmore than about 20% for cell proliferation and more than about 2-fold ofIC₅₀. In embodiments, the nucleic acid may comprise an antisense nucleicacid, siRNA or shRNA. In embodiments, the cell may comprise a cancerstem cell or a neoplastic cell.

In still another embodiment, the compositions and methods of the presentinvention involve a first therapy an inhibitor of miR-192 or miR-215 oran expression construct encoding miR-192 or miR-215, used in combinationwith a second therapy to enhance the effect of the miR-192 or miR-215therapy, or increase the therapeutic effect of the second therapy beingemployed to treat a neoplasm. These compositions would be provided in acombined amount effective to achieve the desired effect, such as thekilling of a cancer cell and/or the inhibition of cellularhyperproliferation. This process may involve contacting the cells withthe miR-192 or miR-215 and the second therapy at the same or differenttime. This may be achieved by contacting the cell with one or morecompositions or pharmacological formulation that includes one or more ofthe agents, or by contacting the cell with two or more distinctcompositions or formulations, wherein one composition provides (1)administering to the subject an effective amount of a nucleic acidmolecule that inhibits expression of miR-192 or miR-215 and/or (2) asecond therapy, in which the inhibition of expression of miR-192 ormiR-215 sensitizes the neoplasm to the second therapy.

The second therapy may comprise administering chemotherapy,radiotherapy, surgical therapy, immunotherapy or gene therapy. In anembodiment, the chemotherapeutic agent is selected from the groupconsisting of a DHFR inhibitor and a TS inhibitor. The chemotherapeuticagents include, but are not limited to, DHFR inhibitors or TS inhibitorssuch as, for example, MTX, pemetrexed, 5-FU, raltitrexed (TDX),nolatrexed, ZD9331, and/or GS7904L. One of ordinary skill wouldrecognize other chemotherapeutic agents useful in this embodiment. It iscontemplated that the combination therapy may be provided in anysuitable manner or under any suitable conditions readily apparent to oneof ordinary skill in the art.

For example, administration of any compound or therapy of the presentinvention to a patient will follow general protocols for theadministration of such compounds, taking into account the toxicity, ifany, of the vector or any protein or other agent. Therefore, in someembodiments there is a step of monitoring toxicity that is attributableto combination therapy. It is expected that the treatment cycles wouldbe repeated as necessary. It also is contemplated that various standardtherapies, as well as surgical intervention, may be applied incombination with the described therapy.

A wide variety of other chemotherapeutic agents may be used inaccordance with the present invention. A “chemotherapeutic agent” isused to connote a compound or composition that is administered in thetreatment of cancer. These agents or drugs are categorized by their modeof activity within a cell, for example, whether and at what stage theyaffect the cell cycle. Alternatively, an agent may be characterizedbased on its ability to directly cross-link DNA, to intercalate intoDNA, or to induce chromosomal and mitotic aberrations by affectingnucleic acid synthesis. Most chemotherapeutic agents fall into thefollowing categories: alkylating agents, antimetabolites, antitumorantibiotics, mitotic inhibitors, and nitrosoureas.

In embodiments, the neoplasm being treated is a form of cancer. Cancersthat may be evaluated by methods and compositions of the inventioninclude any suitable cancer cells known to one of ordinary skill in theart. The inventors have found that the present invention is particularlyuseful in treating cancer cells from the colon or the pancreas,including pancreatic ductal adenocarcinoma. However, other suitablecells include cancer cells of the bladder, blood, bone, bone marrow,brain, breast, cervix, esophagus, gastrointestine, gum, head, kidney,liver, lung, nasopharynx, neck, ovary, prostate, rectum, skin, stomach,testis, tongue, or uterus. Other conditions treatable by thecompositions and methods of the present invention will be readilyapparent to one of ordinary skill in the art.

An inhibitory nucleic acid molecule of the invention, or other negativeregulator of miR-192 or miR-215 may be administered within apharmaceutically-acceptable diluent, carrier, or excipient, in unitdosage form. Conventional pharmaceutical practice may be employed toprovide suitable formulations or compositions to administer thecompounds to patients suffering from a neoplasia. Administration maybegin before the patient is symptomatic. Any appropriate route ofadministration may be employed, for example, administration may beparenteral, intravenous, intraarterial, subcutaneous, intratumoral,intramuscular, intracranial, intraorbital, ophthalmic, intraventricular,intrahepatic, intracapsular, intrathecal, intracisternal,intraperitoneal, intranasal, aerosol, suppository, or oraladministration. Therapeutic formulations and methods for making suchformulations are well known in the art.

The formulations can be administered to human patients intherapeutically effective amounts to provide therapy for a neoplasticdisease or condition. The preferred dosage of inhibitory nucleic acid ofthe invention is likely to depend on such variables as the type andextent of the disorder, the overall health status of the particularpatient, the formulation of the compound excipients, and its route ofadministration.

Therapy may be provided at any suitable location and under any suitableconditions. The duration of the therapy depends on various factorsreadily understood by one of ordinary skill in the art. Drugadministration may also be performed at any suitable interval. Forexample, therapy may be given in predetermined on-and-off intervals asappropriate.

Depending on the type of cancer and its stage of development, thetherapy can be used to slow the spreading of the cancer, to slow thecancer's growth, to kill or arrest cancer cells, to relieve symptomscaused by the cancer, or to prevent cancer. As described herein, ifdesired, treatment with an inhibitory nucleic acid molecule of theinvention may be combined with therapies such as, for example,radiotherapy, surgery, or chemotherapy for the treatment ofproliferative disease.

In another embodiment, a method of diagnosing a neoplasm in a subject isprovided. In this embodiment, the method comprises determining the levelof expression of miR-192 or miR-215.

As described herein, the present invention has identified increases inthe expression of miR-192 or miR-215, and corresponding decreases in theexpression of TS and DHFR that are associated with cellularproliferation. Alterations in the expression level of one or more of thefollowing other markers used to diagnose a neoplasia are alsocontemplated. If desired, alterations in the expression of anycombination of these markers is used to diagnose or characterize aneoplasia as would be readily apparent to one of ordinary skill in theart.

In an embodiment, a subject is diagnosed as having or having apropensity to develop a neoplasia, the method comprising measuringmarkers in a biological sample from a patient, and detecting analteration in the expression of test marker molecules relative to thesequence or expression of a reference molecule. While the followingapproaches describe diagnostic methods featuring a miR-192 or miR-215,the skilled artisan will appreciate that any one or more of the markersset forth above is useful in such diagnostic methods. Increasedexpression of a miR-192 or miR-215 is correlated with neoplasia.Accordingly, the invention provides compositions and methods foridentifying a neoplasia in a subject. The present invention provides anumber of diagnostic assays that are useful for the identification orcharacterization of a neoplasia. Alterations in gene expression aredetected using methods known to the skilled artisan and describedherein. Such information can be used to diagnose a neoplasia.

In an embodiment, diagnostic methods of the invention are used to assaythe expression of miR-192 or miR-215 in a biological sample relative toa reference sample. In one embodiment, the level of miR-192 or miR-215is detected using a nucleic acid probe that specifically binds at leasta portion of miR-192 (SEQ ID NO:1) or miR-215 (SEQ ID NO:9). Exemplarynucleic acid probes that specifically bind miR-192 or miR-215 aredescribed herein. The biological sample can be any sample commonly usedwithin the art, such as, for example, blood, urine, tissue or otherbodily fluid.

In an embodiment, quantitative PCR methods are used to identify anincrease in the expression of miR-192 or miR-215. In another embodiment,PCR methods are used to identify an alteration in the sequence ofmiR-192 or miR-215. The invention provides probes that are capable ofdetecting miR-192 or miR-215. Such probes may be used to hybridize to anucleic acid sequence derived from a patient having a neoplasia. Thespecificity of the probe determines whether the probe hybridizes to anaturally occurring sequence, allelic variants, or other relatedsequences. Hybridization techniques may be used to identify mutationsindicative of a neoplasia or may be used to monitor expression levels ofthese genes.

In embodiments, a measurement of a nucleic acid molecule in a subjectsample may be compared with a diagnostic amount present in a reference.Any significant increase or decrease in the level of test nucleic acidmolecule or polypeptide in the subject sample relative to a referencemay be used to diagnose a neoplasia. Test molecules include any one ormore of markers disclosed herein. In an embodiment, the reference is thelevel of test polypeptide or nucleic acid molecule present in a controlsample obtained from a patient that does not have a neoplasia. Inanother embodiment, the reference is a baseline level of test moleculespresent in a biologic sample derived from a patient prior to, during, orafter treatment for a neoplasia. In yet another embodiment, thereference can be a standardized curve. The subject sample can be anysample commonly used within the art, such as, for example, blood, urine,tissue or other bodily fluid.

In another embodiment, a method of identifying a neoplasm resistant tochemotherapy is provided. In this embodiment, the method comprisesdetermining the level of expression in the neoplasm of miR-192 ormiR-215, and identifying the neoplasm as resistant to therapy if thelevel of miR-192 or miR-215 is elevated, or identifying the neoplasm asnot resistant to therapy if the level of miR-192 or miR-215 is reduced.

In another embodiment, a method of determining whether a neoplasm is acandidate for treatment with a chemotherapeutic agent is provided. Inthis embodiment, the method comprises evaluating the level of expressionof miR-192 or miR-215 and rejecting the candidate if expression of themiR-192 or miR-215 is elevated, or accepting the candidate if theexpression of miR-192 or miR-215 is reduced.

In another embodiment, a kit for analysis of a pathological sample isprovided. Any of the compositions described herein may be comprised inthe kit. In a non-limiting example, reagents for isolating miRNA,labeling miRNA, and/or evaluating a miRNA population using an array,nucleic acid amplification, and/or hybridization can be included in akit, as well as reagents for preparation of samples from blood samples.The kit may further include reagents for creating or synthesizing miRNAprobes. The kits may comprise, in suitable container means, an enzymefor labeling the miRNA by incorporating labeled nucleotides or unlabelednucleotides that are subsequently labeled. In certain aspects, the kitcan include amplification reagents. In other aspects, the kit mayinclude various supports, such as glass, nylon, polymeric beads, and thelike, and/or reagents for coupling any probes and/or target nucleicacids. It may also include one or more buffers, such as a reactionbuffer, labeling buffer, washing buffer, or a hybridization buffer,compounds for preparing the miRNA probes, and components for isolatingmiRNA. Other kits of the invention may include components for making anucleic acid array comprising miRNA, and thus, may include, for example,a solid support. The pathological sample can be any sample commonly usedwithin the art, such as, for example, blood, urine, tissue or otherbodily fluid.

Kits for implementing methods of the invention described herein arespecifically contemplated. In some embodiments, there are kits forpreparing miRNA for multi-labeling and kits for preparing miRNA probesand/or miRNA arrays. In these embodiments, the kit may comprise, insuitable container means, any suitable solvents, buffers, reagents, oradditives known to one of ordinary skill in the art including, but notlimited to, those generally used for manipulating RNA, such asformamide, loading dye, ribonuclease inhibitors, and DNase.

In other embodiments, kits may include an array containing miRNA probes.Such arrays may include, for example, arrays relevant to a particulardiagnostic, therapeutic, or prognostic application. For example, thearray may contain one or more probes that is indicative of a disease orcondition, susceptibility or resistance to a drug or treatment,susceptibility to toxicity from a drug or substance, prognosis, and/orgenetic predisposition to a disease or condition.

For any kit embodiment, including an array, there can be nucleic acidmolecules that contain or can be used to amplify a sequence that is avariant of, identical to or complementary to all or part of any SEQ IDdescribed herein. In certain embodiments, a kit or array of theinvention can contain one or more probes for the miRNAs identified bythe SEQ IDs described herein. Any nucleic acid discussed above may beimplemented as part of a kit.

The components of the kits may be packaged in any suitable manner knownto one of ordinary skill in the art such as, for example, in aqueousmedia or in lyophilized form. The kits of the present invention may alsoinclude a means for containing the nucleic acids, and any other reagentcontainers in close confinement for commercial sale. Such containers mayinclude injection or blow molded plastic containers into which thedesired vials are retained.

In this embodiment, the kits may also include components that facilitateisolation of the labeled miRNA. It may also include components thatpreserve or maintain the miRNA or that protect against its degradation.Such components may be RNAse-free or protect against RNAses. Such kitsgenerally will comprise, in suitable means, distinct containers for eachindividual reagent or solution.

A kit will also include instructions for employing the kit components aswell as the use of any other reagent not included in the kit.Instructions may include variations that can be implemented.

In another embodiment, a method of identifying an agent that inhibitsthe expression or activity of miR-192 or miR-215 is provided. Inembodiments, the method comprises contacting a cell that expresses themiR-192 or miR-215 with an agent, and comparing the expression level ofthe miR-192 or miR-215 in the cell contacted by the agent with theexpression level of the miR-192 or miR-215 in the absence of the agent.According to this embodiment, the agent is an inhibitor of the miR-192or miR-215 if expression of the miR-192 or miR-215 is reduced. In thisembodiment, the test cell may overexpress the miRNA.

Compounds that modulate the expression or activity of a miR-192 ormiR-215 are useful in the methods of the invention for the treatment,prevention, diagnosis and prognostication of a neoplasm or subject. Themethod of the invention may measure a decrease in transcription ofmiR-192 or miR-215 or an alteration in the transcription or translationof the target of miR-192 or miR-215. Any number of methods are availablefor carrying out screening assays to identify such compounds. In anembodiment, the method comprises contacting a cell that expressesmiR-192 or miR-215 with an agent and comparing the level of miR-192 ormiR-215 expression in the cell contacted by the agent with the level ofexpression in a control cell, wherein an agent that decreases theexpression of miR-192 or miR-215 thereby, in combination with asecondary therapy, inhibits a neoplasia. In another embodiment,candidate compounds are identified that specifically bind to and alterthe activity of miR-192 or miR-215 of the invention. Methods of assayingsuch biological activities are known in the art. The efficacy of such acandidate compound is dependent upon its ability to interact withmiR-192 or miR-215. Such an interaction can be readily assayed using anynumber of standard binding techniques and functional assays.

Potential agonists and antagonists of miR-192 or miR-215 include, butare not limited to, organic molecules, peptides, peptide mimetics,polypeptides, nucleic acid molecules, and antibodies that bind to anucleic acid sequence of the invention and thereby inhibit or extinguishits activity. Potential antagonists also include small molecules thatbind to miR-192 or miR-215 thereby preventing binding to cellularmolecules with which the miR-192 or miR-215 normally interacts, suchthat the normal biological activity of miR-192 or miR-215 is reduced orinhibited. Small molecules of the invention preferably have a molecularweight below 2,000 daltons, more preferably between 300 and 1,000daltons, and still more preferably between 400 and 700 daltons. It ispreferred that these small molecules are organic molecules.

The invention also includes novel compounds identified by theabove-described screening assays. These compounds are characterized inone or more appropriate animal models to determine the efficacy of thecompound for the treatment of a neoplasia. Characterization in an animalmodel can also be used to determine the toxicity, side effects, ormechanism of action of treatment with such a compound. Furthermore,novel compounds identified in any of the above-described screeningassays may be used for the treatment of a neoplasia in a subject. Suchcompounds are useful alone or in combination with other conventionaltherapies known in the art.

It is also contemplated that the invention can be used to evaluatedifferences between stages of disease, such as between hyperplasia,neoplasia, precancer and cancer, or between a primary tumor and ametastasized tumor. Moreover, it is contemplated that samples that havedifferences in the activity of certain pathways may also be compared. Itis further contemplated that nucleic acids molecules of the inventioncan be employed in diagnostic and therapeutic methods with respect toany of the above pathways or factors. Thus, in some embodiments of theinvention, a miRNA may be differentially expressed with respect to oneor more of the above pathways or factors. The samples can be any samplecommonly used within the art, such as, for example, blood, urine, tissueor other bodily fluid.

In certain embodiments, miRNA profiles may be generated to evaluate andcorrelate those profiles with pharmacokinetics. For example, miRNAprofiles may be created and evaluated for patient tumor and bloodsamples prior to the patient's being treated or during treatment todetermine if there are miRNAs whose expression correlates with theoutcome of the patient. Identification of differential miRNAs can leadto a diagnostic assay involving them that can be used to evaluate tumorand/or blood samples to determine what drug regimen the patient shouldbe provided. In addition, it can be used to identify or select patientssuitable for a particular clinical trial. If a miRNA profile isdetermined to be correlated with drug efficacy or drug toxicity, thatmay be relevant to whether that patient is an appropriate patient forreceiving the drug or for a particular dosage of the drug. One ofordinary skill in the art would recognize that other samples, such asurine, bodily fluid or tissue, can also be used.

In addition to the above prognostic assay, samples from patients with avariety of diseases can be evaluated to determine if different diseasescan be identified based on blood miRNA levels. A diagnostic assay can becreated based on the profiles that doctors can use to identifyindividuals with a disease or who are at risk to develop a disease. Thesamples may be any sample commonly taken from a patient, such as, forexample, blood, urine or tissue. Alternatively, treatments can bedesigned based on miRNA profiling.

All references mentioned herein are incorporated in their entirety byreference into this application.

It is to be understood and expected that variations in the principles ofthe invention herein disclosed may be made by one skilled in the art andit is intended that such modifications are to be included within thescope of the present invention. The following examples only illustrateparticular ways to use the novel technique of the invention, and shouldnot be construed to limit the scope of the invention in any way.

Example 1 miR-192 Regulates the Expression of DHFR

There are several miRNAs that potentially interact with the 3′-UTRregion of DHFR mRNA. Bioinformatic analysis of the secondary structureof the 3′-UTR of the DHFR mRNA and miRNA binding sites reduced thecandidate miRNAs to a small number. From this analysis, miR-192 wasidentified as a candidate that may regulate DHFR.

FIG. 1 shows the secondary structure of the 3′-UTR of the DHFR mRNA andthe target sequence that interacts with miR-192. To experimentallyconfirm the expression of DHFR was regulated by miR-192, a miR-192precursor was transfected into HCT-116 (wt-p53) cells. A non-specificmiR was used as a negative control. It has been reported that theexpression of DHFR is regulated by miR-24. A DHFR siRNA and miR-24 wereused as positive controls. Over-expression of miR-192 (FIG. 2A) andmiR-24 (FIG. 2B) was confirmed by real time qRTPCR analysis using U6 RNAto normalize the expression.

The expression of DHFR protein was analyzed using Western immunoblotanalysis and the results are shown in FIG. 2C. In this experiment, humancolon cancer cell lines, HCT-116 (wt-p53), cells were transfected withmiR-192 or miR-24. The cells were maintained in McCoy's 5A medium (LifeTechnologies). The media was supplemented with 10% dialyzed fetal bovineserum (Hyclone Laboratories). The cell line were grown at 37° C. in ahumidified incubator with 5% CO₂.

The HCT-116 (wt-p53) cells transfected as follows. The cells (2×10⁵)were plated in 6-well plates and transfected with 100 nmol/L of eithermiR-192 or miR-24 precursors or nonspecific control miR (Ambion) after24 h with Oligofectamine (Invitrogen) according to the manufacturer'sinstructions. Small interfering RNA (siRNA) specific to DHFR (On-Targetplus SMARTpool L-008799-00-0010, human DHFR, NM_(—)000791) (Dharmacon)and transfected with Oligofectamine (Invitrogen) according to themanufacturer's protocols at a final concentration of 100 nmol/L. siRNAspecific to DHFR was used as the positive control. miR-24, a miRNA thatalso targets DHFR (Mishra, et al., “A miR-24 microRNA binding sitepolymorphism in dihydrofolate reductase gene leads to methotrexateresistance: Prof. Nat'l. Acad. Sci. (2007) 104:13513-13518) was alsoused as a positive control.

At 48 hours after transfection with miR-192 or miR-24 precursors ornonspecific control miRNA, the cells were scraped and lysed inradioimmunoprecipitation assay buffer (Sigma). Equal amount of proteinswere resolved by SDS-PAGE on 12% gels by the method of Laemmli,“Cleavage of structured proteins during the assemble of the head ofbacteriophase 74,” Nature (1970) 227:680-685, and transferred topolyvinylidene difluoride membranes (Bio-Rad Laboratories). Themembranes were then blocked by 5% nonfat milk in TBS-0.5% Tween 20 atroom temperature for 1 hour. The primary antibodies used for theanalysis included mouse anti-DHFR monoclonal antibody (mAb; 1:250; BDBiosciences), and mouse anti-a-tubulin mAb (1:1,000; TU-02) (Santa CruzBiotechnology). Horseradish peroxidase-conjugated antibodies againstmouse or rabbit (1:1,000; Santa Cruz Biotechnology) were used as thesecondary antibodies. Protein bands were visualized with achemiluminescence detection system using the Super Signal substrate.

FIG. 2C is the resulting Western immunoblot that shows overexpression ofmiR-192 clearly decreased the expression of DHFR protein (FIG. 2C, lane4). In this image, lane 1 is the control, lane 2 is the cellstransfected with non-specific miR control, lane 3 is the cellstransfected with siRNA specific to DHFR, lane 4 is from cellstransfected with miR-192 and lane 5 is the cells transfected withmiR-24. The results show that the potency of miR-192 (FIG. 2C, lane 4)for decreasing DHFR expression was comparable to miR-24 (FIG. 2C, lane5).

The expression level of DHFR mRNA were analyzed using real time qRT-PCRanalysis. The analysis was performed as follows. Total RNA, includingmiRNA, was isolated from cell lines by using TRIzol reagent (Invitrogen)according to the manufacturer's instructions at 24 h after transfection.cDNA was synthesized with the High Capacity cDNA synthesis kit (AppliedBiosystems) using 2 μg of total RNA as the template and random primers.Real-time qRT-PCR analysis was done on the experimental mRNAs. The PCRprimers and probes for DHFR, and the internal control gene GAPDH werepurchased from Applied Biosystems. qRT-PCR was done on an ABI 7500HTinstrument under the following conditions: 50° C., 2 min of reversetranscription; 95° C., 10 min; 95° C., 15 sec; 60° C., 1 min. Thereaction was done for up to 40 cycles (n=3). GAPDH was used as aninternal standard for normalization.

The results (FIG. 2D) indicated that there was no reduction in DHFR mRNAexpression by miR-192 (lane 4) and miR-24 (lane 5). In FIG. 2D, each barcorresponds to each lane illustrated in FIG. 2C (see above). The resultsdemonstrate that the suppression of DHFR expression was regulated at thetranslational level without the degradation of DHFR mRNA. By contrast,the decreased expression of DHFR by siRNA was clearly caused by mRNAdegradation (lane 3, FIG. 2D).

Example 2 Increased Expression of miR-192 Sensitizes Cells toMethotrexate (MTX) Treatment

This was confirmed by studying cellular proliferation in cellstransfected with miR-192 and treated with MTX. HCT-116 (wt-p53) cellswere plated in 96-well plates at 1×10³ cells/well in triplicate. Theywere transfected with miR-192 precursor (FIG. 3, lane 4), non-specificcontrol miRNA (FIG. 3, lane 2), or siRNA against DHFR (FIG. 3, lane 3)in 100 μl of medium. Twenty-four hours later, MTX in 100 μl medium(final concentration 25 nM) was added, and incubated for 72 hours. Onesample was only transfected with non-specific miR and was not treatedwith MTX (FIG. 3, lane 1). Ten microliters of WST-1 (Roche AppliedScience) was added to each well. After 2 hours of incubation, absorbancewas measured at 450 and 630 nm respectively (n=3). Non-specific controlmiRNA alone was used as a negative control (FIG. 3, lane 1), and siRNAincubation with MTX was used as a positive control (FIG. 3, lane 3).

With equal molar concentration of MTX at 25 nM (IC-10), cellproliferation was reduced by 10% in non specific control miR treatedcells (FIG. 3, lane 2). However, cell proliferation was reduced bynearly 70% in cells transfected with miR-192, demonstrating asynergistic effect in combination with MTX (FIG. 3, lane 4). Bycontrast, cells treated with siRNA against DHFR were inhibited by 55%(FIG. 3, lane 3). The more potent effect of miR-192 plus MTX compared tosiRNA targeting specifically to DHFR suggests that miR-192 may alsotarget additional mRNA targets through imperfect base pairing.

Example 3 miR-192 Inhibits Cell Proliferation of HCT-116 (wt-p53), RKO(wt-p53) and HT-29 (mut-p53) Colon Cancer Cell Lines

To assess the functional significance of miR-192, the impact of miR-192on cellular proliferation was evaluated using HCT-116 (wt-p53), HCT-116(null-p53), RKO (wt-p53) and HT-29 (mut-p53) colon cancer cell lines.The human colon cancer cell lines HCT-116 (wt-p53) and HCT-116(null-p53) were provided by Prof. Bert Vogelstein (The Johns HopkinsUniversity) and were maintained in McCoy's 5A medium (LifeTechnologies). The other two human colon cancer cell lines, RKO (wt-p53)and HT-29 (mut-p53), were obtained from the American Type CultureCollection. The HT-29 (mut-p53) cell line has a missense mutation incodon 273 of p53 resulting in an Argto-His substitution. RKO (wt-p53)and HT-29 (mut-p53) cells were maintained in Eagle's MEM and Iscove'sModified Dulbecco's Medium at the American Type Culture Collection,respectively. All media were supplemented with 10% dialyzed fetal bovineserum (Hyclone Laboratories). All cell lines were grown at 37° C. in ahumidified incubator with 5% CO₂. The cell lines were transfected asdescribed above (see Examples 1 and 2).

The cellular proliferation was evaluated as follows. HCT-116 (wt-p53)(FIG. 4A), HCT-116 (null-p53) (FIG. 4C), RKO (wt-p53) (FIG. 4B), andHT-29 cells (FIG. 4D) were plated in 96-well plates in triplicate at1×10³ cells per well after transfection with miR-192 precursor(illustrated as —▴—) or non-specific control miRNA (illustrated as —▪—).Cells were cultured for 24, 48, 72, or 96 hours. The absorbance at 450and 630 nm was measured after incubation with 10 μl of WST-1 for 2hours. A non-specific miR was used as a negative control.

The results (see FIG. 4) show that the overexpression of miR-192 cansuppress cellular proliferation in HCT-116 (wt-p53) cells by over 55%(n=3) (FIG. 4A) and RKO (wt-p53) cells by 48% (n=3) (FIG. 4B), with lessimpact on HCT-116 (null-p53) (15%, n=3) (FIG. 4C) and HT-29 cell lines(24%, n=3) (FIG. 4D). By contrast, the non-specific control miR has noeffect on cellular proliferation, indicating that this effect caused bymiR-192 is highly specific. The results clearly showed that the effectof miR-192 on the inhibition of cellular proliferation is more potent incolon cancer cells containing wild type p53, further indicating thatmiR-192's function depends on the status of p53.

Example 4

To determine whether the miR-192's impact on cellular proliferation wasrelated to cell cycle control, the effect on cell cycle control wasanalyzed by flow cytometry using HCT-116 (wt-p53) (FIG. 5A) and HCT-116(null-p53) (FIG. 5B) cells transfected with non-specific control miR(left line graph) or miR-192 (right line graph). This experiment wasperformed as follows. The HCT-116 (wt-p53) and HCT-116 (null-p53) cellslines as described above (see Example 3) were transfected with miR-192precursor and the non-specific control miRNA described as above (seeExamples 1 and 2). At 36 hours after transfection, the cells wereharvested and resuspended at 0.5-1×10⁵ cells/ml in modified Krishanbuffer containing 0.1% sodium citrate and 0.3% NP-40 and kept at 4° C.Before being analyzed by flow cytometry, cells were treated with 0.02mg/ml RNase H and stained with 0.05 mg/ml propidium iodide (Sigma).

In the HCT-116 (wt-p53) cells transfected with non-specific miR had thefollowing cell cycle profile G1: 38.78%, G2: 17.46%, S: 43.76%, G1/S:0.89%, and G2/S: 0.40%. The HCT-116 (wt-p53) cells transfected withmiR-192 had the follow cell cycle profile G1: 40.84%, G2: 44.71%, S:14.45%, G1/S: 2.83%, and G2/S: 3.09%. In the HCT-116 (null) cellstransfected with non-specific miR had the following cell cycle profileG1: 40.15%, G2: 13.33%, S: 46.52%, G1/S: 0.86%, and G2/S: 0.29%. TheHCT-116 (null) cells transfected with miR-192 had the follow cell cycleprofile G1: 35.65%, G2: 19.06%, S: 45.29%, G1/S: 0.79% and G2/S: 0.42%.

The results (FIG. 5) show that miR-192 induces both G1 (>2-fold) and G2arrest (>3-fold) in HCT-116 (wt-p53) cells (FIG. 5A). By contrast, thiseffect has not been observed in HCT-116 (null-p53) cells (FIG. 5B). Thecell cycle analysis data is highly consistent with the cellproliferation results that the function of miR-192 is dependent on thepresence of wild type p53 for cell cycle control.

Example 5 miR-192 Expression is Dependent on p53

To further analyze the cell cycle control genes involved in miR-192overexpression, a number of cell cycle control genes were analyzed (p53,p21, Bax, E2F3, Rb). HCT-116 (wt-p53) cells (FIG. 6A) and RKO (wt-p53)cells (FIG. 6B), as described above (see Example 3), were transfectedwith non-specific miR (FIG. 6A, lane 2; and FIG. 6B., lane 1), DHFRsiRNA (FIG. 6A, lane 3; and FIG. 6B., lane 2) and miR-192 (FIG. 6A, lane4; and FIG. 6B., lane 3) according to the procedure discussed above (seeExamples 1 and 2). Non-transfected HCT-116 (wt-p53) was used as anegative control (FIG. 6A, lane 1). Alpha-tubulin was used as a proteinloading control. At 48 hours after transfection, the cells were scrapedand lysed in RIPA buffer (Sigma). Equal amount of proteins were resolvedby SDS-PAGE on 12% gels by the method of Laemmli, and transferred topolyvinylidene fluoride membranes (BIO-RAD Laboratories). The membraneswere then blocked by 5% nonfat milk in TBS-T (Tris-buffered saline and0.5% Tween-20) at room temperature for 1 hour. The primary antibodiesused for the analysis included mouse anti-p53 mAb (1:1000, DO-1) (SantaCruz Biotechnology), mouse anti-p21 mAb (1:1000, F-5) (Santa CruzBiotechnology), and mouse anti-α-tubulin mAb (1:1000, TU-02) (Santa CruzBiotechnology). Horseradish peroxidase-conjugated antibodies againstmouse or rabbit (1:1000) (Santa Cruz Biotechnology) were used as thesecondary antibodies. Protein bands were visualized with achemiluminescence detection system using the Super Signal substrate.

FIG. 6 shows the results of Western immunoblot analysis in HCT-116(wt-p53) 10 cells (FIG. 6A) and RKO (wt-p53) cells (FIG. 6B). miR-192increased the expression of the p53 protein (FIG. 6A, lane 4) over10-fold and p21 10-fold. By contrast, siRNA against DHFR (FIG. 6A, lane3) did not cause an increase in expression of p53 and p21. Theexpression of Bax was not altered by miR-192. Similar results wereobtained for RKO (wt-p53) cells (FIG. 6B, lane 3 (miR-192), lane 1(non-specific miR) and lane 2 (siRNA of DHFR)).

Example 6

The expression of miR-192 on the expression of E2F3 and Rb in HCT-116(wt-p53) cells was also analyzed. In these experiments, HVT-116 (wt-p53)cells, as described above (see Example 3) were transfected withnon-specific miR (FIG. 7, lane 2), DHFR siRNA (FIG. 7, lane 3) ormiR-192 (FIG. 7, lane 4) according to the procedure described above (seeExamples 1 and 2). One HCT-116 (wt-p53) sample was not transfected, andused as a negative control (FIG. 7, lane 1). Western immunoblot analysiswas performed according to the procedure described above (see Example5), except that rabbit anti-E2F-3 polyclonal antibody (1:1000, C-18)(Santa Cruz Biotechnology) was used.

The results (FIG. 7) indicated that miR-192 caused a decreasedexpression of E2F3 and Rb (FIG. 7, lane 4). The results furtherconfirmed the notion that the function of miR-192 is clearly dependenton the status of wild type p53. It has been well characterized that theinduction of the p53 dependent cell cycle check point control gene p21is the key to trigger cell cycle arrest at both the G1 and G2 phase. Theexpression of the proapoptotic protein Bax was not altered, suggestingthat the reduced proliferation may not be due to increased apoptosis.miR-192 overexpression also caused a slight decrease in the expressionof E2F3 and Rb (FIG. 7), some of the key regulators of the G2/Mtransition. The decreased expression of E2F3 and Rb may be partiallyresponsible for both the G1/S and G2/M arrest caused by miR-192overexpression.

Example 7

With over-expression of miR-192 by transfection, both HCT-116 (wt-p53)and RKO (wt-p53) cells undergo cell cycle arrest at the G1 and G2 phaseleading to decreased cellular proliferation. Bioinformatic analysis alsoreveals that there is a putative p53 binding site in the miR-192promoter region.

To confirm this direct regulatory relationship, the followingexperiments were performed. First, the expression of the p53 protein wasinduced by treatment with MTX in HCT-116 (wt-p53) cells, RKO (wt-p53)cells, HCT-116 (null-p53) cells and HT-29 (mut-p53) cells (see Examples1 and 2 for description of cell lines). Western immunoblot using mouseanti-p53 mAb (1:1000, DO-1) (Santa Cruz Biotechnology) according to theprocedure discussed in Example 4 demonstrated that MTX treatment inducedp53 protein expression (FIG. 8A).

Thereafter, the expression of endogenous mature miR-192 was analyzed byreal time qRT-PCT analysis using an internal control RNU6B as aninternal standard for normalization. The cDNA synthesis was carried outwith the High Capacity cDNA synthesis kit (Applied Biosystems) using 10ng of total RNA as template. The miRNA sequence-specific RT-PCR primersfor miR-192 and endogenous control RNU6B were purchased from Ambion.Real-time quantitative reverse transcription-PCR (qRT-PCR) analysis wascarried out using Applied Biosystems 7500 Real-Time PCR System. The PCRmaster mix containing TaqMan 2× Universal PCR Master Mix (No AmperaseUNG), 10+ TaqMan assay and RT products in 20 μl volume were processed asfollows: 95° C. for 10 minutes, and then 95° C. for 15 seconds, 60° C.for 60 seconds for up to 40 cycles (n=3). Signal was collected at theendpoint of every cycle. The gene expression ΔC_(T) values of miRNAsfrom each sample were calculated by normalizing with internal controlRNU6B and relative quantitation values were plotted.

The results are illustrated in FIG. 8B where an open bar indicates anMTX(−) sample, and a dashed bar indicates an MTX(+) sample. The qRT-PCT(FIG. 8B) shows that induction of p53 by MTX caused a significantincrease of miR-192 expression. By contrast, MTX treatment in HCT-116(null-p53) and HT-29 (mut-p53) cells did not cause any change in theexpression of miR-192 (FIG. 8B). These results suggest that theendogenous expression of miR-192 depends on the wild type p53.

Example 8

The promoter site of miR-192 contains a well conserved p53 bindingsequence. FIG. 9A is a schematic diagram showing the position of themiR-192 promoter (over 3 kb) relative to the location of the miR-192precursor on chromosome 11. To experimentally confirm a directinteraction between the p53 protein and the miR-192 promoter, chromatinimmunoprecipitation-qRT-PCT (ChIP-qPCR) analysis was used to isolatep53-bound chromosome DNA. p21, a known cell cycle regulatortranscriptionally regulated by p53, was used as a positive control.Mouse monoclonal antibody (DO-1) against p53 (Santa Cruz Biotechnology)was used for immunoprecipitation of the p53 binding complex. Non-relatedantibody α-tubulin (TU-02, Santa Cruz Biotechnology) was used as anegative control. Immunoprecipitation was performed based on themanufacturer's protocols of Active Motif. The primer sequences for themiR-192 promoter and the p21 promoter are listed as follows:

miR-192 promoter (forward primer): (SEQ ID NO: 4)5′-AGCACCTCCCATGTCACC-3′ miR-192 promoter (reverse primer):(SEQ ID NO: 5) 5′-CAAGGCAGAGCCAGAGC-3′ p21 promoter (forward primer):(SEQ ID NO: 6) 5′-GCTGGTGGCTATTTTGTCCTTGGGC-3′p21 promoter (reverse primer): (SEQ ID NO: 7)5′-AGAATCTGACTCCCAGCACACACTC-3′

The isolated p53 specific binding DNA was PCR amplified using primerswhich span the predicted p53 binding sites of the miR-192 promoter orthe positive control p21 promoter transcriptionally regulated by p53protein.

FIG. 9 shows the immunoprecipitation qPCR analysis of the predictedmiR-192 promoter with conserved p53 binding sequence. FIG. 9B shows thatthe p53 protein directly interacts with the miR-192 promoter based onChIP-qPCR analysis with a 4-fold enriched signal of with p53 specificmonoclonal antibody compared to the non-specific antibody control DNA.This data establishes the existence of a conserved p53 binding site atthe promoter region of miR-192. The binding sequence is5′-cgccatgcctxxxggccttgccc-3′ (SEQ ID NO:25), with a 3-base-pair gaprepresented by “x”. This suggests that miR-192, like miR-34, is anothermiRNA that is involved in the p53 tumor suppressor network.

Luciferase reporter assay was used to determine the transcriptionalactivation of conserved p53-binding promoter of miR-192. pGL3-Basicpromoterless luciferase reporter plasmid (Promega) was used in thisstudy. Double-stranded DNA oligonucleotides of conserved p53-bindingsequence of miR-192 was synthesized and annealed and cloned upstream offirefly luciferase in the pGL3-Basic plasmid (miR-192-pGL3). Thep53-binding site oligonucleotide (bold) contains MluI at the 5′-end andBglII sequence at the 3′-end(5′-ACGCGTCCCATGTCACCACCAGGGGTCGCCATGCCTCCTGGCCTTGCCCAGCAAG ATCT-3′)(SEQ ID NO:8). Control vector and miR-192-pGL3 vector were transfectedinto both HCT-116 (wt-p53) and HCT-116 (null-p53) cells. To furtherinduce p53 expression, transfected HCT-116 (wt-p53) and HCT-116(null-p53) cells were also treated with 5 μmol/L 5-fluorouracil for 24hours. The promoter activity of each construct was quantified by dualluciferase assay (Promega) 24 hours after transfection. Fireflyluciferase temperature for 1 hour. The primary antibodies used for theanalysis included mouse anti-DHFR monoclonal antibody (mAb; 1:250) (BDBiosciences), mouse anti-p53 mAb (1:1,000; DO-1) (Santa CruzBiotechnology), mouse antip21mAb (1:1,000; F-5) (Santa CruzBiotechnology), and mouse anti-a-tubulin mAb (1:1,000; TU-02) (SantaCruz Biotechnology). Horseradish peroxidase-conjugated antibodiesagainst mouse or rabbit (1:1,000) (Santa Cruz Biotechnology) were usedas the secondary antibodies. Protein bands were visualized with achemiluminescence detection system using the Super Signal substrate.

The conserved p-53 binding site at the promoter region of miR-192 canactivate luciferase expression only in HCT-116 (wt-p53) cells. Theactivation was further enhanced by induced p53 expression in HCT(wt-p53) cells treated with 5-fluorouracil. By contrast, the inductionof luciferase activity was completely absent from the HCT-116 (null-p53)cells. This evidences that miR-192, like miR-34, is another miRNA thatis involved in the p53 tumor suppressor network.

p53 is one of the most frequent altered tumor suppressor gene incolorectal cancer. The potential function of multiple miRNAs involved inp53 tumor suppressor network is to provide the p53 with greaterflexibility in rapidly responding to different growth condition changes,by perhaps having unique miRNAs (e.g. miR-34, miR-192) mediate theregulation of the key mRNA targets. miR-192 was one of the miRNAs withreduced expression in a large cohort of colon cancer patient samples,further supporting the potential impact and clinical relevance ofmiR-192 in colon cancer. The decrease or loss of the suppressivefunction of miR-192 in colon cancer may be an important factor relatedto cell cycle control and chemosensitivity to anti-folate based therapy.

Example 1-8 establish that miR-192 is directly involved in theregulation of a key anticancer target DHFR. The expression and functionof the miR-192 is largely dependent on the presence of functional wildtype p53. Thus, miR-192 may be used as a novel therapeutic option fortreating cancer via an effective delivery system either alone or incombination with anti-folate compounds.

Example 9 miR-215 Inhibits Cell Proliferation of HCT-116(wt-p53) and U-2OS Cells

HCT-116 (wt-p53), HCT-116 (null-p53), U-2 OS, and MG63 cells (2×10⁵)were plated in six-well plates, and transfected with 100 nM of eithermiR-215, miR-192 precursors or non-specific control miRNA (Ambion) after24 h by Oligofectamine (Invitrogen) according to the manufacturer'sprotocols. siRNA specific to TS (Mishra, et al., “AmiR-24 microRNAbinding site polymorphism in dihydrofolate reductase gene leads tomethotrexate resistance,” Prof. Nat'l. Acad. Sci. (2007)104:13513-13518) and siRNA specific to DHFR (ON-TARGET plus SMARTpoolL-008799-00-0010, human DHFR, NM_(—)000791) were purchased fromDharmacon and transfected with Oligofectamine (Invitrogen) at a finalconcentration of 100 nM. siRNA specific to TS or DHFR were used as thepositive controls. miR-192, an miRNA that targets DHFR, was also used asthe positive control.

Total RNA, including miRNA, was isolated from cell lines at 24 h aftertransfection, or from Stem cell lines or snap frozen tissues by usingTRIzol reagent (Invitrogen) according to the manufacturer'sinstructions.

Real-time qRT-PCR confirmed the increased expression of miR-215 in thetransfected cells. cDNA synthesis was carried out with the High CapacitycDNA synthesis kit (Applied Biosystems) using 10 ng of total RNA astemplate. The miRNA sequence-specific RT-PCR primers for miR-215 andendogenous control RNU6B were purchased from Ambion. Real-timequantitative reverse transcription-PCR (qRT-PCR) analysis was carriedout using Applied Biosystems 7500 Real-Time PCR System. The PCR mastermix containing TaqMan 2× Universal PCR Master Mix (No Amperase UNG), 10×TaqMan assay and RT products in 20 μl volume were processed as follows:95° C. for 10 min, and then 95° C. for 15 sec, 60° C. for 60 sec for upto 40 cycles (n=3). Signal was collected at the endpoint of every cycle.The gene expression ΔC_(T) values of miRNAs from each sample werecalculated by normalizing with internal control RNU6B and relativequantitation values were plotted.

Cell proliferation assays were performed on the transduced cells.HCT-116 (wt-p53), HCT-116 (null-p53), U-2 OS, and MG63 cells were platedin 96-well plates in triplicate at 1×10³ cells/well after transfectionwith miR-215 precursor or non-specific control miRNA. Cells werecultured for 24, 48, 72, 96 h. The absorbance at 450 and 630 nm wasmeasured after incubation with 10 μl of WST-1 for 2 h. A remarkableinhibition of cell proliferation was observed in either HCT-116 (wt-p53)or U-2 OS cells compared with the non-specific miRNA control, and thereduction in HCT-116 (wt-p53) or U-2 OS cells were approximately 40% or58% at day 5 respectively (FIG. 10).

miR-215 Induces G2-Arrest in HCT-116 (Wt-p53) and U-2 OS Cells.

To investigate the mechanism by which miR-215 suppressed cellproliferation in HCT-116 (wt-p53) and U-2 OS cells, the impact ofmiR-215 on cell cycle control was analyzed by flow cytometry at 36 hafter transfection. HCT-116 (wt-p53), HCT-116 (null-p53), U-2 OS andMG63 cells were transfected with miR-215 precursor and the non-specificcontrol miRNA described as above. At 36 h after transfection, cells wereharvested and resuspended at 0.5-1×10⁵ cells/ml in modified Krishanbuffer containing 0.1% sodium citrate and 0.3% NP-40 and kept at 4° C.Before analysis by flow cytometry, cells were treated with 0.02 mg/mlRNase H and stained with 0.05 mg/ml propidium iodide (Sigma).

In miR-215 transduced U-2 OS cells, the proportion of cells in theG2-phase was higher and the proportion of cells in the S-phase was lowerthan that in control cells (18.50% vs 29.69%; 52.08% vs 41.24%; FIG.11). The G2/S ratio was increased in miR-215 transfected cells (0.35 vs0.72; >2-fold). The results showed that miR-215 causes increasedaccumulation of cells at G2-phase, whereas cells in S-phase decrease.Similar results were observed in transfected I-WT-116 (wt-p53) cells.

miR-215 Increases the Expression of Cell Cycle Control Genes p53 andp21.

p53 and p21, a downstream target of the p53 pathway of growth control,are reported to block cells at G2 checkpoint mainly through inhibitingactivity of Cdc2, the cyclin-dependent kinase that normally drives cellsinto mitosis, which is the ultimate target of pathways that mediaterapid arrest in G2 in response to DNA damage. To further analyze themechanism of cell proliferation inhibition by miR-215, we transfectedmiR-215 precursor into HCT-116 (wt-p53) and U-2 OS cells, and evaluatedthe levels of cell cycle control genes p53 and p21 by western immunoblotanalysis.

At 48 h after transfection with miR-215, miR-192 precursors ornon-specific control miRNA, the cells were scraped and lysed in RIPAbuffer (Sigma). Equal amounts of proteins were resolved by SDS-PAGE on12% gels by the method of Laemmli, and transferred to polyvinylidenefluoride membranes (BIO-RAD Laboratories). The membranes were thenblocked by 5% nonfat milk in TBS-T (Tris-buffered saline and 0.5%Tween-20) at room temperature for 1 h. The primary antibodies used forthe analysis included mouse anti-TS mAb (1:400, Millipore), mouseanti-DHFR mAb (1:250, BD Bioscience), mouse anti-p53 mAb (1:1000, DO-1),mouse anti-p21 mAb (1:1000, F-5), and mouse anti-α-tubulin mAb (1:1000,TU-02) purchased from Santa Cruz Biotechnology. Horseradishperoxidase-conjugated antibodies against mouse (1:1000, Santa CruzBiotechnology) were used as the secondary antibodies. Protein bands werevisualized with a chemiluminescence detection system using the SuperSignal substrate (BIO-RAD).

Over-expression of miR-215 led to a significant increase of the p53 andp21 protein in both HCT-116 (wt-p53) and U-2 OS cells. (FIG. 12). Theresults indicated that miR-215 contributed to the inhibition of cellproliferation at least partially by the induction of G2-arrest inHCT-116(wt-p53) and U-2 OS cells through over-expression ofG2-checkpoint genes p53 and p21.

Example 10 DHFR and TS are the Direct Targets of miR-215

Using the Sanger database (microrna.sanger.ac.uk) TS and DHFR wereidentified as putative targets of miR-215 (FIG. 13). At 48 hours aftertransfection, proteins were extracted from miR-215-transfected cells andfrom control cells and changes in TS or DHFR protein levels weredetermined by western immunoblot analysis (FIG. 14). Oligofectaminealone and non-specific miRNA were used as the negative controls.miR-192, which had been observed to down-regulate DHFR, was used as apositive control of DHFR down-regulation. Over-expression of miR-192 andmiR-215 was confirmed by real time qRT-PCR analysis using U6 RNA tonormalize the expression. Introduction of miR-192 or miR-215 clearlydecreased TS or DHFR protein levels (FIG. 14, lane 3 and 4). Expressionlevels of TS or DHFR mRNA were analyzed using real time qRT-PCR analysis(FIG. 15). The results indicated that there was no reduction in TS orDHFR mRNA expression by miR-215 (column 3) and miR-192 (column 4). Thus,the suppression of TS or DHFR expression was regulated at thetranslational level without the degradation of TS or DHFR mRNA.

To confirm whether miR-215 directly targets TS or DHFR, plasmids wereconstructed containing the fragments of the 3′UTR of TS or DHFR in thedownstream region of firefly luciferase. The pMIR-REPORT LuciferasemiRNA Expression Reporter Vector (Ambion) was used to determine thetargets of miR-215. Double stranded DNA oligonucleotides containing themiR-215 binding sequence (wt-miR-215) or mismatch sequence (mut-miR-215)in 3′UTR of TS or DHFR mRNA and the HindIII and SpeI restriction siteoverhangs were synthesized (IDT, Coralville, Iowa) and annealed andcloned downstream of firefly luciferase in the pMIR-REPORT plasmid. Thesequences of these synthesized oligonucleotides are provided below. The3′UTR of TS includes two miR-215 binding sites: one is located at 84-104bp, one is located at 216-236 bp.

198-247 bp of 3′UTR of TS mRNA Forward-wt-miR-215 (SEQ ID NO: 14)5′-CTAGTAGTTAACTCCCTGAGGGTATCTGACAATGCTGAGGTTATGA ACAAAGTGA-3′Reverse-wt-miR-215 (SEQ ID NO: 15)5′-AGCTTCACTTTGTTCATAACCTCAGCATTGTCAGATACCCTCAGGG AGTTAACTA-3′Forward-mut-miR-215 (SEQ ID NO: 16) 5′-CTAGTAGTTAACTCCCTGAGGGTATATCACGATG T TGATATCAC GA ACAAAGTGA-3′ Reverse-mut-miR-215 (SEQ ID NO: 17)5′-AGCTTCACTTTGTTCGTGATATCAACATCGTGATATACCCTCAGGG AGTTAACTA-3′62-115 bp of 3′UTR of TS mRNA Forward-wt-miR-215-2 (SEQ ID NO: 18)5′-CTAGTAGTTCTTTTTGCTCTAAAAGAAAAAGGAACTAGGTCAAAAA TCTGTCCGA-3′Reverse-wt-miR-215-2 (SEQ ID NO: 19)5′-AGCTTCGGACAGATTTTTGACCTAGTTCCTTTTTCTTTTAGAGCAA AAAGAACTA-3′519-578 bp of 3′UTR of DHFR mRNA Forward-wt-miR-215 (SEQ ID NO: 20)5′-CTAGTAATTTCAGTGAAAGCAGTGTATTTGCTAGGTCATACCAGAAATCATCAATTGAGGTACGGA-3′ Reverse-wt-miR-215 (SEQ ID NO: 21)5′-AGCTTCCGTACCTCAATTGATGATTTCTGGTATGACCTAGCAAATACACTGCTTTCACTGAAATTA-3′ Forward-mut-miR-215 (SEQ ID NO: 22)5′-CTAGTAATTTCAGTGAAAGCAGTGT GCTTGC GATATG ATACCAGAAATCATCAATTGAGGTACGGA-3′ Reverse-mut-miR-215 (SEQ ID NO: 23)5′-AGCTTCCGTACCTCAATTGATGATTTCTGGTATCATATCGCAAGCACACTGCTTTCACTGAAATTA-3′

The constructs were transiently transfected into HCT-116 (wt-p53) cellsalone (control) or together with miR-215 precursor. Twenty-four hoursbefore transfection, HCT116 (wt-p53) and HCT 116 (null-p53) cells wereplated in the 96-well plates at 1.5×10⁴ cells each well in triplicate.pMIR-REPORT constructs (100 ng) together with ing of Renilla luciferaseplasmid phRL-SV40 (Promega, Madison, Wis.) were transfected byLipofectamine 2000 (Invitrogen, Carlsbad, Calif.) according to theprotocols provided by the manufacturer. Thirty hours after transfection,cells were lysated and luciferase activity was measured by thedual-luciferase reporter assay system (Promega, Madison, Wis.) accordingto the instructions. Firefly luciferase activity for each condition wasnormalized by dividing to Renilla internal control and then compared topMIR-REPORT.

As shown in FIG. 16, there was a significant decrease of luciferaseactivity compared to the vector alone. These data demonstrated that TSor DHFR were the direct targets of miR-215, and suggested that theinhibition of cell proliferation by miR-215 was partially due to thedown-regulation of these two enzymes.

Example 11 Reduced Chemosensitivity to TDX and MTX by Overexpression ofmiR-215

TS and DHFR are the major targets of cancer chemotherapy in the clinic.TDX, the third-generation TS inhibitor, is an active agent in thetreatment of human colon and breast cancer. Inhibitors of DHFR, such asMTX, are widely used in the treatment of human leukemia, osteosarcomaand choriocarcinoma. Increased DHFR protein levels are reported to beassociated with drug resistance, and low tumor expression levels of TShave also been linked with improved outcome for colon cancer patientstreated with 5-FU chemotherapy. However, Yamauchi et al observed thatMTX has the highest activity at the time when DNA synthesis, DHFRactivity, DHFR content, and DHFR mRNA content increased and the lowestactivity at the time when they decreased.

In this study, we confirmed miR-215 decreased the expression of TS andDHFR protein, we then test if miR-215 can change the sensitivity of TDXor MTX in the HCT-116 (wt-p53) or U-2 OS cells.

HCT-116 (wt-p53) cells were plated in 96-well plates at 1×10³ cells/wellin triplicate and were transfected with miR-215 precursor, non-specificcontrol miRNA, or siRNA against TS or DHFR in 100 μl of medium.Twenty-four hours later, TDX or MTX in 100 μl medium ranged from 10-200nM was added, and incubated for 72 hours. To measure viable cells, 10 μlof WST-1 (Roche Applied Science) was added to each well. After 2 hoursincubation, absorbance was measured at 450 and 630 nm respectively(n=3). Non-specific control miRNA alone was used as a negative control,and siRNAs incubation with TDX or MTX were used as the positivecontrols.

FIG. 17A shows that the IC₅₀ of TDX in HCT-116(wt-p53) cells transfectedwith miR-215 was 98.7 nM, whereas in the negative control was 18.6 nM,in the positive control was 8.5 nM. FIG. 8B showed that 83.6% oftransduced U-2 OS cells still alive at 200 nM of MTX. The IC₅₀ of MTX inthe negative control was 49.7 nM, whereas 37.3 nM in the positivecontrol. These results indicated that down-regulation of TS or DHFRprotein by the siRNA specific against TS or DHFR indeed increased thesensitivity of TDX or MTX in the colon cancer or osteosarcoma celllines, whereas even though miR-215 also down-regulated the expressionlevels of TS or DHFR, it did not increase the chemosensitivity of TDX orMTX compared to the non-specific miRNA control. TDX or MTX areconsidered to be more effective on the cells in the S-phase. The cellcycle data showed that siRNAs specific against TS or DHFR did notdecrease the cells in the S-phase (FIG. 11), whereas the cells in theS-phase were reduced in the miR-215 transfected cells.

Over-Expression of miR-215 in Human Colon Cancer Stem Cells MayContribute to the Low Sensitivity to TDX and MTX.

Cancer stem cells also named cancer initiating cells, exhibit low rateof division and proliferation in their niche that help them to avoidchemotherapy and radiation. To determine whether miR-215 expressioninfluences the growth and chemosensitivity in cancer stem cells, miR-215levels in colon cancer stem cells were measured using real-time qRT-PCR.Expression of miR-215 was determined to be greater than 2-fold higher inthe colon cancer stem cells than in the controls (FIG. 18).

Example 12 Clinical Human Colon Cancers Show Decreased miR-215Expression

miR-215 expression in 22 paired human colon cancer and counterpartnormal tissues was analyzed by real-time qRT-PCR (FIG. 19). Eighteen of22 colon cancer samples (81.8%) showed decreased miR-215 level (P<0.05).

From the description and examples provided above, one of ordinary skillin the art would appreciate the application of miR-192 and miR-215 invarious diagnostic tools and treatments.

Example 13

It is contemplated that miR-192 and miR-215 would be altered in asubject simultaneously according to the methods discussed above, or thatthe inhibitory molecule would comprise a combination of miR-192 andmiR-215. When both are altered, it is expected that there would begreater than the effect observed with either miR-192 or miR-215, orwould have a synergistic effect. For example, it is expected that ifmiR-192 and miR-215 are both inhibited in a subject, then thesensitivity to a chemotherapeutic agent would be increased, and thatthis increase would be greater than the increase in sensitivity observedwith miR-192 or miR-215. It is further expected that if miR-192 andmiR-215 are upregulated together, then the decrease in cellproliferation would be greater than observed with miR-192 or miR-215.

What is claimed is:
 1. A method of modulating expression of a componentof a cell, comprising contacting the cell with a nucleic acid comprisingan inhibitory nucleic acid having a sequence selected from the groupconsisting of SEQ ID NOs:1, 2, 3, 9, 10, 25, a complementary sequencethereof, a variant thereof on a fragment thereof in an amount sufficientto modulate the cellular component.
 2. The method of claim 1, whereinthe nucleic acid is an antisense nucleic acid.
 3. The method of claim 1,wherein the nucleic acid is an siRNA or an shRNA.
 4. The method of claim1, wherein the cellular component is selected from the group consistingof miR-192 and miR-215.
 5. The method of claim 1, wherein the cellularcomponent is p21 or p53.
 6. The method of claim 1, wherein the cellularComponent is regulated by p53.
 7. The method of claim 1, wherein thecellular component is DHFR or TS.
 8. A method of increasingproliferation of a cell, comprising contacting the cell with a nucleicacid complementary to at least a portion of a sequence selected from thegroup consisting of SEQ ID NOs:1, 2, 3, 9, 10, 25 and an antisensesequence of SEQ ID NO:25, in an amount effective to increaseproliferation of the cell.
 9. A method of increasing the sensitivity ofa cell to a chemotherapeutic agent, comprising contacting the cell witha nucleic acid complementary to a sequence selected from the groupconsisting of SEQ ID NOs:1, 2, 3, 9, 10, 25 and an antisense sequence ofSEQ ID NO:25, in an amount effective to sensitize the cell to thechemotherapeutic agent.
 10. The method of claim 9, wherein thechemotherapeutic agent is selected from methotrexate, fluorouracil(5-FU), and ralitrexed.
 11. A method of increasing the sensitivity of acell to radiation, comprising contacting the cell with a nucleic acidcomplementary to at least a portion of a sequence selected from thegroup consisting of SEQ ID NOs:1, 2, 3, 9, 10, 25 and an antisensesequence of SEQ ID NO:25, in an amount effective to sensitize the cellto radiation.
 12. The method of any one of claims 8 to 11, wherein thenucleic acid is an antisense nucleic acid.
 13. The method of any one ofclaims 8 to 11, wherein the nucleic acid is an siRNA or an shRNA. 14.The method of any one of claims 8 to 11, wherein the cell is a cancerstem cell.
 15. The method of any one of claims 8 to 11, wherein the cellis a neoplastic cell.
 16. A method of treating a neoplasm in a subject,comprising administering to the subject an effective amount of aninhibitory molecule that inhibits expression of miR-192 or miR-215, anda second therapy, wherein inhibition of expression of miR-192 or miR-215sensitizes the neoplasm to the second therapy.
 17. The method of claim16, wherein the second therapy comprises administering achemotherapeutic agent.
 18. The method of claim 17, wherein thechemotherapeutic agent is selected from methotrexate, fluorouracil(5-FU), and ralitrexed.
 19. The method of claim 16, wherein the secondtherapy comprises administering radiation to the subject.
 20. The methodof any one of claims 16 to 19, wherein the neoplasm is cancer.
 21. Themethod of claim 20, wherein the cancer is selected from the groupconsisting of colon cancer, pancreatic cancer, lung cancer, breastcancer, cervical cancer, gastric cancer, kidney cancer, leukemia, livercancer, lymphoma, ovarian cancer, prostate cancer, rectal cancer,sarcoma, skin cancer, testicular cancer, and uterine cancer.
 22. Amethod of diagnosing a neoplasm in a subject comprising determining thelevel of expression of miR-192 or miR-215.
 23. A method of identifying aneoplasm resistant to chemotherapy comprising determining the level ofexpression in the neoplasm of a microRNA (miRNA) selected from the groupconsisting of miR-192 and miR-215, and identifying the neoplasm asresistant to therapy if the level of the miRNA is elevated.
 24. A methodof determining whether a neoplasm is a candidate for treatment with achemotherapeutic agent comprising evaluating the level of expression ofa microRNA (miRNA) selected from the group consisting of miR-192 andmiR-215, and rejecting the candidate if expression of the miRNA iselevated.
 25. A kit for analysis of a pathological sample, the kitcomprising in a suitable container a hybridization reagent for,determining the level of a miRNA selected from the group consisting ofmiR-192 or miR-215.
 26. The kit of claim 25, wherein the hybridizationreagent comprises a hybridization probe.
 27. The kit of claim 25,wherein the hybridization reagent comprises amplification primers.
 28. Amethod of identifying an agent that inhibits expression of a microRNA(miRNA) selected-from the group consisting of miR-192 and miR-215comprising contacting a cell that expresses the miRNA with an agent; andcomparing an expression level of the miRNA in the cell contacted by theagent with an expression level of the miRNA in the absence of the agent,wherein the agent is an inhibitor of the miRNA if expression of themiRNA is reduced.
 29. The method of claim 28, wherein the test celloverexpresses the miRNA.
 30. An isolated inhibitory molecule comprisinga nucleic acid, wherein the nucleic acid consists essentially of asequence selected from the group consisting of SEQ ID NO:9, SEQ IDNO:25, a complementary sequence of SEQ ID NO:9, a complementary sequenceof SEQ ID NO:25, fragments thereof and variants thereof.
 31. Theisolated inhibitory molecule, whenever the sequence is selected from thegroup consisting of the complementary sequence of SEQ ID NO:9, thecomplementary sequence of SEQ ID NO:25, fragments thereof and variantsthereof.
 32. The isolated inhibitory molecule, wherein the sequence isSEQ ID NO:25 or a complementary sequence of SEQ ID:25.